GenomeRef

GRCr8: A new rat reference assembly is released!

noreply@blogger.com (grc) — Fri, 02 Feb 2024 22:55:00 +0000

GRCr8 (GCA_036323735.1), the latest version of the rat reference genome assembly, is now available. GRCr8 is an evolution of mRatBN7.2 (GCA_015227675.2), the Vertebrate Genomes Project-generated rat assembly that was the first reference for this species to be adopted by the GRC for stewardship. mRatBN7.2 was an assembly of a Brown Norway (BN) male rat from the same colony at the Medical College of Wisconsin that supplied the female rat used in the 2004 RGSC_v3.4 assembly (AABR00000000.3/GCF_000001895.1). While the assembly of mRatBN7.2 was a substantial improvement over prior versions (https://pubmed.ncbi.nlm.nih.gov/37214860/), advances in sequencing technology and assembly and curation methods since its release in 2020 have for the first time resulted in the GRC releasing a new de novo assembly as a reference update instead of curating issues in the prior version.

GRCr8 was generated by Dr. Peter Doris (University of Texas Health Science Center at Houston) with colleagues Theodore Kalbfleisch (University of Kentucky) and Melissa Smith (University of Louisville) in the NHGRI-funded “Inbred Rat Genomes Project”. The assembly is based on PacBio HiFi sequences from a BN/NHsdMcwi male rat. The assembly was gap filled using contigs from the PacBio CLR reads produced for mRatBN7.2. Additional short read genomic sequence from a BN/NHsdMcwi rat in the Hybrid Rat Diversity Program at the Medical College of Wisconsin were used for assembly polishing. In addition to yielding a consensus quality score (QV) of 59.5, GRCr8 addresses structural limitations of mRatBN7.2. The genome size is increased from 2.63Gb to 2.81Gb, largely because of incorporation of genomic regions that show structural expansion. For example, chrY has increased from 18Mb in mRatBN7.2 to 60Mb in GRCr8. Accompanying multi-tissue single molecule transcript information (PacBio IsoSeq) is available for this assembly (BioProject: PRJNA1027884). These data extend the scope of rat transcript diversity and will inform gene expression in newly incorporated regions of the genome.

The GRCr8 assembly has been submitted to the INSDC, making it available through GenBank, ENA and DDBJ. It will subsequently be annotated by groups such as RefSeq and Ensembl, after which it will be available on genome browsers at various resources, including the Rat Genome Database, NCBI, Ensembl, and UCSC.

GRCh38.p14 is now released!

noreply@blogger.com (grc) — Mon, 09 May 2022 14:29:00 +0000

GRCh38.p14 (GCA_000001405.29/GCF_000001405.40), the latest update to the human reference assembly, has been released! It adds 69 new patch scaffolds, 51 of which are FIX patches that update sequences on the GRCh38 reference chromosomes or alternate loci, while 18 are NOVEL patches, providing new alternate representations for complex genomic regions that are inadequately represented by a single sequence. Two previously released FIX patches were also updated. With this release, the reference assembly contains a total of 250 patch scaffolds (164 FIX, 90 NOVEL).

30 of the patches included in this release include genome updates made in support of the MANE project, a joint NCBI-EBI effort to produce a minimal set of matching RefSeq and Ensembl transcripts of protein coding genes, creating a matched pair of transcripts but retaining their respective identifiers. Read more about MANE effort in their recent Nature publication. The corresponding patch updates to the reference assembly involved changes addressing normal human variation as well as correcting errors in the underlying component sequences.

Of the 53 FIX patches in GRCh38.p14, 23 of these correct errors in individual assembly component sequences, resulting in updates to 12 gene representations (Table 1). 20 are variation-related updates, 12 of which provide the coding allele for 13 polymorphic pseudogenes that are non-coding on the corresponding GRCh38 chromosomes (Table 2). Additionally, 2 provided sequence updates at chromosomal loci where it's unclear if the GRCh38 sequence is in error or a rare haplotype. Patch scaffolds in GRCh38.p14 close 6 gaps in the reference assembly, and extend sequence into one other gap. 4 of the closed gaps are located within chromosomes, while the remaining 2 patch scaffolds closed "pre-telomeric" gaps, extending the sequence of the chromosome into the telomeric repeats.

Table 1. Gene representations updated on FIX patches addressing assembly component problems.

Table 2. Coding alleles of polymorphic pseudogenes updated by FIX patches addressing genomic variation.

An example of an important FIX patch in this release is an update to APOB, one of the genes the American College of Medical Genetics and Genomics recommends for reporting of incidental findings in clinical exome and genome sequencing. The patch scaffold provided in GRCh38.p14 represents the common allele.

There are 18 NOVEL patches in this release, providing alternate sequence representations of chromosomal sequences, including 9 genes (Table 3). Other NOVEL patches represent inversion and insertion haplotypes relative to the corresponding chromosomal region.

Table 3. Genes with alternate sequence representation on GRCh38.p14 NOVEL patches.

Shown below is an example of an update to PRDM9, a medically important gene in which naturally occuring allelic variation regulates the activity of meiotic recombination hotspots. The original GRCh38 release represents the relatively rare "B" allele on chromosome 5. With the release of GRCh38.p14, a NOVEL patch scaffold has been added to the assembly (MU273356.1/NW_025791779.1) to provide additional representation for the sequence of the more common "A" allele.

Figure 1. PRDM9 allele representation in GRCh38.p14. Top: Alignment of PRDM9 "A" (NM_001310214.3) and "B" (NM_001376900.1) allele transcripts to chromosome 5. The chromosome sequence represents the "B" allele. The red circles and arrows highlight mismatches in the alignment of the "A" allele. Bottom: Alignment of Alignment of PRDM9 "A" and "B" allele transcripts to the NOVEL patch added in GRCh38.p14. The patch represents the "A" allele. The red circle highlights mismatches in the alignment of the "B" allele.

Notably, 9 of the NOVEL patches used clone sequence generated by Evan Eichler's lab as part of a published study of the evolution and population diversity of human-specific segmental duplications.The GRC also used sequences generated by the Eichler lab to create a FIX patch to improve a GRCh38 chromosome 5 alternate locus scaffold (KI270897.1/NT_187651.1) representing the haplotype from the CHM1 hydatidiform mole at the hypervariable SMA locus. Informed by CHM1 Bionano optical map data, the GRC provided a FIX patch (MU273354.1/NW_025791777.1) that corrects component order and adds sequence from several newly sequenced CHM1 BAC clones to the alternate locus scaffold.

Figure 2. A FIX patch corrects the sequence path of the GRCh38 alt locus scaffold providing representation of the CHM1 haplotype for the SMA region on chromosome 5. Top: Tiling path of component clones in the alt loci scaffold. Middle: Tiling path of component clones in the FIX patch scaffold. Blue outline: clones excluded from fix scaffold. Green outline: clones added to fix scaffold. Magenta outline: clones from alt scaffold retained in fix scaffold. Black: sequence gap. Bottom: Alignment of fix patch scaffold path to CHM1 Bionano optical map, demonstrating concordance.

This patch release also extends GRC efforts to identify and exclude problematic sequences, such as false redundancies and contamination, from the reference assembly. The companion BED file available from GenBank that identifies such regions and can be used as a mask to exclude them from analyses, has now been updated. The latest updates reflect curation done in response to reports from GRCh38 analyses performed by the Genome In a Bottle (GIAB) and Telomere-to-Telomere (T2T) consortia. In addition to the chromosome 21p regions previously reported, the file provides coordinates for 7 other regions in which the sequence falsely duplicates other sequence found in the assembly.

We are grateful to our community collaborators for the sequences and analyses that contributed to the updates in GRCh38.p14. Please alert the GRC if you have specific assembly issues to report, or contact us for any questions or feedback. We'd love to hear from you!

One of these things doesn't belong: efforts to exclude problematic sequences in GRCh38

noreply@blogger.com (grc) — Wed, 21 Jul 2021 11:52:00 +0000

Since the release of GRCh38, the GRC has received a number of user reports alerting us to a potential false duplication involving chr 21p and 21q. Users noted that reads were aligning to both regions in GRCh38, but not GRCh37/hg19, resulting in a decreased mapping score and difficulties in variant calling throughout. Additionally, user analyses involving Multiplex Ligation-dependent Probe Amplification (MLPA), a technique for gene copy number detection, and exome studies indicated potential false duplications. The implicated regions contained several genes, including CBS (Gene ID: 875), U2AF1 (Gene ID: 7307) and KCNE1B (Gene ID: 3753). The GRC has investigated the matter and concurs that the GRCh38 assembly contains sequence on the short arm of chr 21 that should be excluded from analyses. Read on to learn more about this issue, as well as some recently detected non-human contamination in GRCh38, and ways you can find and avoid these sequences in your analyses.

The short arm of human chromosome 21, like that of the four other human acrocentric chromosomes, is where genes associated with rDNA synthesis are localized, and is characterized by highly repetitive heterochromatic sequence. The repetitive nature of these sequences, coupled with limitations in sequencing technology, have until recently made the representation of these regions in genome assemblies very difficult.

As a consequence, the GRCh37 representation of the chromosome 21 p-arm contained only 11 clone sequences. Seven were clones from the HSA21-specific BAC library CHORI-507 that had previously been experimentally localized to 21p (PMID: 17895424). In an effort to add additional sequence to this repetitive region, 23 additional components were added to 21p for GRCh38, including 18 additional CHORI-507 clones, 4 RPCI-11 clones, and 1 ABC9 fosmid. Admixture mapping localized some of these clones to this region.

In response to the user reports, the GRC re-reviewed the sequences added to 21p in GRCh38. Haploid CHM13hTERT Illumina reads generated by The McDonnell Genome Institute were aligned to GRCh38 by NCBI, and evaluated for read mapping and coverage. This analysis supported the user reports, suggesting that 5 of the newly added CHORI-507 clones (FP565260.4, CU639417.17, FP236240.8, FP475955.4 and CU633980.13) were actually redundant with sequences on chr 21q, and thus represented false duplications in GRCh38.

The GRC has now removed these sequences from the files that it uses to generate the reference assembly. However, we cannot remove them from the GRCh38 assembly without triggering the next major release of the human assembly. In order to help users recognize these regions and avoid them in their analyses, we have produced a masking file to be used as a companion to GRCh38. This BED file is available from the GenBank FTP site: https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/001/405/GCF_000001405.39_GRCh38.p13/GRCh38_major_release_seqs_for_alignment_pipelines/GCA_000001405.15_GRCh38_GRC_exclusions.bed. This file provides the assembly coordinates of the 5 clones incorrectly localized to chr 21p. The Genome in a Bottle Consortium recently posted a preprint demonstrating that using this masking file greatly improves variant calling accuracy in the affected genes (https://doi.org/10.1101/2021.06.07.444885).

In addition to these sequences, the file also includes 2 other assembly scaffolds that were found, after the release of GRCh38, to be contaminated with non-human sequence. These include a chr Un scaffold (KI270752.1/NT_187507.1), whose sole component (AF065393.1) is now known to represent sequence from Chinese hamster (PMID:30486838), likely derived from the human-hamster CHO cell line that was the clone source, and an alternate loci scaffold (KI270825.1/NT_187580.1) whose non-anchor component (AC225822.3) was shown to be chimeric. In AC225822.3, the first 25,375 bases are human sequence matching GRCh38 chr 10 reference component and alternate scaffold anchor sequence AL391421.27, while the rest match Acidithiobacillus thiooxidans sequences from multiple WGS projects (PMID:32398145). Although all sequences in the reference assembly are screened for foreign contamination, these two were not detected at the time of release (2014). Prompted by these findings, the GRC has more recently re-screened the assembly with updated contamination databases and has not detected additional issues. As these two scaffolds are not human sequence, very few reads are likely to map well to them, but users may still want to make note of them in their analyses.

In total, the contamination represents ~800 Kb, or 0.02% of the total sequence length. GRCh38 remains an extremely high quality reference assembly. Nonetheless, the GRC remains committed to addressing assembly errors and making sure it serves as the most reliable analysis substrate possible. Check out our website to see other genomic regions under review. We welcome your feedback and reports of newly discovered issues! In the future, we plan to update the masking file with any new regions as identified and reviewed by the GRC.

Fig. A

Fig. A: Aligned CHM13hTERT Illumina reads viewed in Integrative Genomics Viewer (IGV). The panes labeled 'Original' were reads aligned prior to redundant sequence masking and the panes labeled 'Fixed' are reads aligned after redundant sequence masking.

The top two panes show reads aligned to the valid U2AF1 locus in GRCh38 (NC_000021.9:43,091,000-43,110,000 of 21q) and the bottom two panes show reads aligned to the falsely duplicated (pseudo region) region of GRCh38 (NC_000021.9:6,480,000-6,500,000 of 21p).

In IGV, sequence reads that align to 2 places in the Reference (whether it is correct or not), yield poor/ambiguous alignments, indicated by clear, unshaded reads. This is shown by both the 'Original hg38 U2AF1' and the 'Original hg38 pseudo region' pane.

Following the masking of the known, duplicated region introduced in GRCh38, the aligned reads in the 'Fixed hg38 U2AF1 gene' pane are shaded grey, meaning they have good mapping scores to that region. And there are no reads mapping to the 'Fixed hg38 pseudo region' because the duplicated sequence is masked in the Fixed hg38 file.

Fig. B

Fig. B: Aligned reads to 21q region that has false duplication on 21p in GRCh38 before masking. Note the BAC clone boundary where alignment of falsely duplicated region in 21p starts. This duplication involves the CBS gene (Gene ID: 875).

Fig. C

Fig. C: Aligned reads to 21p region falsely duplicated (in Fig. B). You can see ambiguous read alignment and the falsely duplicated CBS gene annotated in the gene track.

Fig. D

Fig. D: Alignment of BAC FP236240.8 (redundant BAC added to 21p for GRCh38) to the corresponding valid region on 21q. Note the redundant BAC alignment to the region (bottom pane) and the valid read alignment depth (shown in middle pane). Since the region was falsely duplicated, read alignment in the region of redundant BAC alignment is poor.

A New Rat Genome Assembly Sparks Membership of Rat and RGD in the Genome Research Consortium

noreply@blogger.com (grc) — Mon, 30 Nov 2020 22:42:00 +0000

The Rat Genome Database RGD is very pleased to announce the release of mRatBN7.1, the new rat genome assembly! The mRatBN7 assembly, generated by the Darwin Tree of Life Project at the Wellcome Sanger Institute, is significantly improved over the Rnor6.0 and previous assemblies. mRatBN7 was derived from a male BN/NHsdMcwi rat that is a direct descendent from the female BN rat previously sequenced. The new BN rat reference genome was generated using multiple technologies including PacBio long reads, 10X linked reads, Bionano maps and Arima Hi-C. Its quality is a substantial improvement compared to any of the previous assemblies, with just 175 scaffolds and having an N50 >135Mb and 756 contigs with N50 >29Mb, resulting in a contiguity similar to the human or mouse reference assemblies. The assembly has been submitted to the International Nucleotide Sequence Database Consortium (INSDC), and the initial GenBank record for it is now available at https://www.ncbi.nlm.nih.gov/assembly/GCA_015227675.1. Genome annotation, i.e. the assignment of gene positions and prediction of new genes and other genomic elements, will be generated by both NCBI and Ensembl. We are also pleased to announce that Rat and the mRatBN7 assembly have been accepted into the Genome Reference Consortium (GRC) and the RGD has been approved to represent the rat research community and participate in the ongoing work of curating the assembly. RGD will work closely with curators from the GRC, with the International Rat Omics Consortium (IROC), a grassroots community of rat genomics researchers, and the rat research community to identify any candidate regions for focused genome curation. Stay tuned for the appearance of rat on the GRC website!

Hi-C 2D map of mRatBN7.1 generated with HiGlass

GRCm39: the new mouse reference genome assembly

noreply@blogger.com (grc) — Wed, 22 Jul 2020 13:15:00 +0000

The GRC is pleased to announce the release of GRCm39 (GCA_000001635.9), the latest version of the mouse reference genome assembly.

GRCm39 is the first coordinate-changing update to the mouse reference since the 2012 release of GRCm38. More than 400 reported issues were resolved in the production of the new assembly, which also incorporates the sequence edits released as scaffolds in the six GRCm38 patch releases.

The new reference assembly exhibits substantial improvements in contiguity. As shown in Fig 1, the scaffold N50 has increased by 95% to 106.1 Mb in GRCm39, and 1.9 Mb of non-N bases were added to the assembly. The gap count has been nearly cut in half, with the total gap length reduced by 4.5 Mb. The decrease in gap length reflects in part the use of optical map data to size the remaining gaps wherever possible, replacing many of the default 50 kb gaps found in GRCm38. Sequences used for gap closures included clones, GRC-constructed contigs, as well as contigs from the C57BL/6J long-read based assembly ASM377452v2.

Figure 1: GRCm39 Assembly Statistics

As in prior assembly versions, the GRCm39 chromosome sequences continue to represent the C57BL/6J strain. However, the alternate loci scaffolds that provided additional strain representations for highly variant genomic regions in GRCm38 and MGSCv37, have been removed from the assembly. The relatively low usage of these scaffolds, coupled with a growing number of high quality strain-specific genome assemblies available in public sequence databases, such as those generated by the Mouse Genomes Project, has reduced the need for the inclusion of these sequences in the reference genome assembly. Although no longer affiliated with the reference assembly, these sequences remain available in the INSDC databases (GenBank/ENA/DDBJ).

The new reference assembly will be annotated by GENCODE and RefSeq in the coming months. An in-depth transcript alignment analysis of a pre-release version of the GRCm39 assembly, presented at the 2019 IMGC meeting, demonstrated that there is improved representation for more than 50 genes. A list of these genes is provided in our earlier blog post. The GRC will provide a complete list of genes improved in GRCm39 as the annotation effort progresses.

Notable curation activities represented in the new assembly, but not in previous patch releases, include the targeted update of more than 1,500 individual bases at which the GRCm38 allele representation was erroneous or an unsupported C57BL/6J variant, a substantial retiling of the chr X pseudo-autosomal region (PAR) that provides representation for several genes missing from GRCm38 (Fig 2), removal of a false triplication involving the Duxbl locus, and correction of a 16 Mb inversion at the proximal end of chromosome 14.

Figure 2. Genes in GRCm39 chr X PAR

The GRC wishes to thank the many members of the mouse community who have reported assembly issues, and contributed their time, expertise, and data to assist in curation efforts. Updates to the GRC website will be made to reflect the new assembly. With the release of GRCm39, the GRC's curation of the mouse genome reference assembly will be limited to the resolution of community reported problems. We encourage you to contact the GRC for additional information on the curation of assembly regions of interest. You can also subscribe to grc-announce email list to receive email notification for all GRC assembly updates.

ZFIN and the GRC: Supporting the zebrafish reference genome assembly

noreply@blogger.com (grc) — Mon, 08 Jun 2020 17:33:00 +0000

ZFIN is a member of The Genome Reference Consortium (GRC), an international collaboration consisting of NCBI, the Wellcome Trust Sanger Institute, the McDonnell Genome Institute at Washington University, the European Bioinformatics Institute (EBI) and ZFIN. This consortium is tasked with ensuring that the reference assemblies for human, mouse, zebrafish and chicken are updated and improved through new data and analysis from genome centers and the research community.

The zebrafish-specific GRC webpage (Fig. 1) provides an overview of the zebrafish genome, including an ideogram of the latest zebrafish assembly (GRCz11) that highlights the location of alternate loci scaffolds, downloadable files for the current public assemby and the tiling path files reflecting the latest assembly edits, as well as links to genome assembly data and genome regions under review. Zebrafish genome issues (Fig. 2), such as sequencing errors, gaps, and path problems, can be browsed at the chromosome level, filtered by problem type or status or searched by gene, location, clone name or accession.

If you come across what you suspect is a problem in the build in the course of your research, visit the GRC website to search the list of genome issues and if it has yet to be reported, select the "Report an Issue" tab in the header (Fig. 3) to report information about the potential problem in the build. Be as complete as possible and provide location, flanking sequences and a description of the issue. Genome annotators will evaluate the region, determine if an update to the genome is needed and submit data to create a new tiling path to improve the build with an update or "patch".

We welcome your feedback!

Figure 1

Figure 2

Figure 3

Readying the release of GRCm39

noreply@blogger.com (grc) — Thu, 23 May 2019 13:25:00 +0000

GRCm38, the current mouse reference assembly, whose chromosomes represent the C57BL/6J strain, supports a broad range of research activities. Despite being one of the highest quality mammalian genome assemblies ever produced, it still has more than 600 gaps and includes sub-optimal representations for some genes. To address these issues and provide the murine research community with an improved substrate for their work, the GRC has been applying new technologies, such as optical/genome mapping, and using new sequence resources to curate an update to the reference genome assembly. The public release of the updated assembly, GRCm39, is planned for the end of 2019/early 2020.

Since the 2012 release of GRCm38, the last coordinate-changing update to the mouse reference, the GRC has provided 6 publicly accessible minor assembly updates, the last of which (GRCm38.p6) was released in September, 2017. These non-coordinate changing assembly versions, known as patch releases, cumulatively include 65 fix patches (chromosome path changes) and 9 novel patches (alternate representations of chromosome sequences, derived from other strains). In GRCm39, these fix patches will be incorporated into the chromosomes, and the novel patches will persist as alternate loci.

In preparation for the release of GRCm39, the GRC also analyzed a non-public updated version of the reference assembly. In the production of this updated assembly, 322 reported genome issues were resolved, 70% of which addressed gaps and problems with the sequence of underlying genomic clone components. The review evaluated the value and impact of the released patches and subsequent unreleased genome updates and assessed the need for additional work prior to the GRCm39 release.

Analyses of this assembly, known informally as "GRCm38B", reveal the removal of about 200 Kb over-expanded sequence found in GRCm38, and the addition of 254 new components, of which 95 are contigs assembled from WGS reads (PRJNA51977). Due to the curation effort, GRCm38B has fewer gaps, and increased contig and scaffold N50s in comparison to GRCm38 (Table 1).

Furthermore, an analysis of mouse RefSeq transcripts aligned to GRCm38B demonstrates the improved representation for at least 50 genes (Table 2).

One such example of improved gene representation in GRCm38B is shown in Figure 1. In GRCm38, an assembly gap at chromosome 4 nt 99,842,111, between components BX324127.8 and CU326395.5, results in a partial representation of Efcab7 (EF-hand calcium binding domain 7). In GRCm38B, sequences from MF597759.1 (a GRC-assembled contig of Illumina reads) closes this gap and provides the exons missing from Efcab7 transcript NM_145549.1.

Figure 1 Top: Incomplete representation of Efcab7 gene in GRCm38 due to an assembly gap. Middle: The gap is closed in GRCm38B with MF597759.1. It also provides complete representation of Efcab7. Bottom: Efcab7-mRNA partial representation in GRCm38 and complete representation in GRCm38B.

Based on GRCm38B analyses, 5 GRCm39 chromosomes will be comprised of a single scaffold (Chr. 11, 12, 15, 16, 18), 11 will be built from 2 scaffolds and the remaining 5 from more than 2 scaffolds.

In the months leading up to the GRCm39 release, the GRC will continue to curate additional genome issues. Sequences from the recently published C57BL/6J long-read based assembly ASM377452v2 are providing new resources for the update or closure of assembly gaps and correction of sequencing errors. Additionally, we are investigating individual bases at which the GRCm38 sequence differs from all 17 strain-specific genome assemblies (Mouse Genome Project) with the aim of correcting confirmed erroneous bases.

Upon the release of GRCm39, the GRC's curation of the mouse genome reference assembly will be limited to the resolution of community reported problems.

You can browse the status of GRC curation activities at our website, and we encourage you to contact the GRC for additional information on the curation of assembly regions of interest. Updates to the timeline for the GRCm39 release will be provided on the Mouse Genome Overview webpage. You can also subscribe to grc-announce email list to receive email notification for all GRC assembly updates .

Shining a light on human acrocentric p-arms

noreply@blogger.com (grc) — Tue, 26 Mar 2019 14:18:00 +0000

The GRC is excited to announce that representations for the p-arms of the human acrocentric chromosomes can now be found in the GRCh38.p13 patch update of the reference genome, thanks to work done in Brian McStay's lab. These sequences are included on the following scaffolds: ML143366.1, ML143367.1, ML143372.1, ML143377.1, and ML143380.1.

The p-arms of the human acrocentric chromosomes HSA13-15, 21 and 22 each bear ribosomal gene arrays (Figure 1) termed nucleolar organiser regions (NORs). These are the most transcriptionally active regions of the genome and direct formation of nucleoli, the largest structures in the nuclei of all human cells. Research on these critical genomic regions is hampered by the fact that acrocentric p-arms are not included in human genome drafts. They are both internally highly repetitive and share a strikingly similar sequence content, making them recalcitrant to standard sequencing approaches. Despite these issues, Brian McStay's lab previously described a collection of sequenced cosmid and BAC clones that allowed them build a reasonable consensus for sequences both immediately proximal and distal to NORs (Floutsakou et al. 2013. Genome Res 23:2003-12). Proximal sequences are almost entirely segmentally duplicated, similar to regions bordering centromeres. In contrast, the distal sequence is predominantly unique to the acrocentric p-arms. Their interphase localisation, open chromatin structure and transcriptionally active state, point to a role in nucleolar biology and prioritise their inclusion in a future genome draft (for discussion see McStay. 2016 Genes Dev. 30:1598-610).

The McStay lab subsequently developed a workflow that has enabled them to determine the NOR distal sequence, the Distal Junction (DJ) from all five acrocentric chromosomes and from an additional two versions of HSA21, ~3 Mb in all. A panel of mono-chromosomal somatic cell hybrids, mouse A9 cells containing individual human chromosomes, allowed them to sequence one chromosome at a time. Sequencing was performed by combining sequence capture with PacBio SMRT sequencing. Pre-capture libraries (typically in the range of 4-6 kb) were prepared from each hybrid line. Capture was performed using oligonucleotide libraries designed using their original consensus. Circular consensus sequencing (CCS) of post-capture libraries generated so called reads of insert (ROIs) each with high sequence accuracy. This allowed the McStay group to assemble sequence contigs from the NOR distal region of each chromosome, regardless of the presence of repetitive sequences such as satellite DNA.

Their analysis of these sequences confirms sequence and presumably functional conservation between the acrocentrics. It also provides evidence for non-homologous exchanges between them. It's anticipated that extension of sequence contigs towards the telomeres will uncover increased structural variation between the acrocentric chromosomes.

Figure 1. FISH experiment showing the relative locations of the rDNA array and distal junctions on the p-arms of the human acrocentric chromosomes.

GRCh38.p13 has been released

noreply@blogger.com (grc) — Wed, 20 Mar 2019 18:37:00 +0000

The GRC is pleased to announce that GRCh38.p13 is now available! This release adds 45 new scaffolds: 43 FIX patches and 2 NOVEL patches. The FIX patch scaffolds provide assembly corrections while the NOVEL patch scaffolds deliver new alternate sequence representations. A valuable contribution to this patch release comes in the addition of the Nucleolus Organiser Region (NOR) sequences for the short arms of the acrocentric chromosomes (13, 14, 15, 21, and 22) as provided by Brian McStay's group (PMID: 23990606). The NOR additions will be discussed in detail in a separate blog.

With access to an ever-increasing pool of high quality, long-read human assembly data, the GRC has been able to utilise this in GRCh38.p13 to address genome issues that have until now persisted due either to lack of data, or complexity. Much of the data added in this patch is derived from the CHM1 human haploid hydatidiform mole assembly (GCA_001297185.2). Originally produced as part of an assembly comparison analysis (see PMID: 28396521), the assembly was recently Pilon corrected and re-submitted to GenBank by the McDonnell Genome Institute at the Washington University, a GRC center, with the specific aim of improving the base pair accuracy for use of its sequences in improving the Human Genome Reference.

In GRCh38.p13, a total of 28 assembly gaps have been closed. These updates, together with sequences added to correct 5 clone errors, add more than 0.5 Mb of unique data to the assembly. The majority of unique sequences added in this release come from contigs that are components of WGS assemblies derived from PacBio sequence reads, such as the CHM1 assembly mentioned above. However, genomic clone libraries still play an important role in assembly curation. In this release BAC clones from human cell line (CHM1htert) have provided complete, single haplotype representations of clinically important regions such as Prader-Willi on chromosome 15, and CT47 on chromosome X.

The CT47 cancer/testis antigen located on human Xq24 is organized as an array of 4.8 kb tandemly repeated units. Due to the repetitive nature of the sequence involved, coupled with the limitations of the technologies available at the time, the representation of the CT47 gene cluster in GRCh37 and GRCh38 was problematic. The region is gapped, and the flanking clones are from different haplotypes. As a consequence, the representation of the cluster in these assemblies was incomplete and biologically unsound, representing an indeterminate number of gene copies (Figure 1, top).
Studies have indicated that this polymorphic array is highly variable between haplotypes and ranges from 4 to 17 copies in length. Long-read sequencing of genomic clones has now captured the complete CT47 cluster as a single haplotype. The fix patch (ML143381.1) included in the GRCh38.p13 release now provides a contiguous and validated representation of the CT47 genomic region. This patch closes the assembly gap with sequences from BAC clone AC275592.1 (CH17-182I12) which contains a complete, 7 copy representation of the CT47 array (Figure 1, bottom). Note that this update reduces the number of CT47 genes represented as compared to GRCh37 and GRCh38.

Figure 1 Top: CT47 region in GRCh38. Incomplete representation of CT47 gene cluster in GRCh38 due to an assembly gap. Bottom: CT47 fix patch in GRCh38.p13. The gap is closed and a complete representation of CT47 cluster is provided.

Optical mapping technology has been used to confirm the copy number for the CT47 array is accurate for the haploid CHM1tert sample (Figure 2), from which the clone library was derived.

Figure 2: AC275592.1 alignment to CHM1 Bionano optical map.

As more data becomes available using the latest technologies the GRC is able to utilise this in order to continually to update and improve the reference genome. If you have questions about this process, let us know.

You can download the GRCh38.p13 assembly, including the alignments of the patches to GRCh38, from the GenBank FTP.

GRCg6: Curation of the chicken reference genome assembly transfers to the GRC

noreply@blogger.com (grc) — Fri, 23 Feb 2018 19:34:00 +0000

The GRC announces the release of the latest chicken reference genome assembly, GRCg6.

The chicken reference assembly defines a standard upon which other avian whole genome studies are based. Providing the best representation of the chicken genome is essential for facilitating continued progress in understanding and improving human health as this species serves as a model organism similar to mouse, zebrafish and other vertebrates.

The chicken reference genome project began as an international research collaboration coordinated by the McDonnell Genome Institute with past funding from the National Institutes of Health (NIH) and U.S. Department of Agriculture whose shared goals were to determine the sequence of the chicken chromosomes and annotate all possible chicken genes. The initial genome reference was completed and published in Nature in 2004 and has since evolved in quality. The reference experienced a major upgrade in 2016, termed Gallus_gallus-5.0 (GCA_000002315.3), as a result of long read sequencing technology and added transcriptome data. In 2017, responsibility for the management of chicken genome assembly updates transferred to the Genome Reference Consortium (GRC). The generation of additional sequence coverage comprised of long read data, in particular average read lengths of 12kb, as well as improvements to de novo assembly algorithms has resulted in another upgrade, GRCg6, that has now been released for immediate community use. Manual annotation of de novo assembled contigs that have been integrated with finished BAC clones have produced an assembly with superior metrics of quality, such as N50 contig size of 18Mb and much lower gap counts.

Visit the chicken homepage at the GRC website for assembly notifications, report assembly issues or contact us with questions.

The GRCg6 assembly will be available in all major genome browsers, and will be annotated by both the NCBI eukaryotic genome annotation pipeline and Ensembl.

* Photo courtesy of Dr. Jerry Dodgson

New technique closes gaps in GRCm38.p6

noreply@blogger.com (grc) — Fri, 09 Feb 2018 13:42:00 +0000

Ongoing efforts to close gaps and to correct clone problems remaining in the GRCm38 mouse reference assembly have proved difficult. The available clone library resources have been exhausted, and the remaining gaps are recalcitrant to cloning, with either no clones available or gap-spanning clones deleted for the expected genomic sequence. The GRC has previously used contigs from publicly available whole genome shotgun assemblies to provide sequence at some of these gaps, and in some cases have been able to close gaps entirely with this approach. Nonetheless, several hundred sequence gaps, many of which are known to contain genes, remain.

With the release of 17 strain-specific genome assemblies from the Mouse Genomes Project, the GRC evaluated alignments between C57BL/6NJ, the most closely related strain, and the GRCm38 reference (C57BL/6J). This evaluation found genes missing from the reference assembly to be present in the new strain assembly. Utilising the C57BL/6J read set (PRJNA51977) deposited in GenBank by the Broad Institute, and used in the production of the C57BL/6J ALLPATHS WGS assembly GCA_000185105.2, the Genome Reference Consortium’s sought to generate local assemblies from these reads that could be used for curation of the GRCm38 reference. The read set was initially aligned to the C57BL/6NJ assembly using bwa-mem. Once completed, reads aligning to the C57BL/6NJ assembly corresponding to GRCh38 gaps and the location of clone-assembly problems in the GRCm38 reference were identified and subsequently assembled using the Geneious software platform (version 10.1.3). The resulting assembly BAMs were then loaded into GAP5 for manual curation. The assembled WGS contigs were then submitted to GenBank.

The patch release GRCm38.p6 addresses 20 regions with these newly created and submitted sequences. These contigs fix and improve representation for several genes, examples of which are shown in Table 1 and Figure 1.

Issue number	Issue type	GenBank_ID	RefSeq_ID	Gene (Gene ID)
MG-3656	Gap	KZ289093.1	NW_019168532.1	Dgkk (331374)
MG-3683	Gap	KZ289094.1	NW_019168533.1	933416I08Rik (71159)
MG-3686	Gap	KZ289095.1	NW_019168534.1	Cylc1 (67407)
MG-104	Gap	KZ289092.1	NW_019168531.1	Atg4a (666468)
MG-3700	Gap	KZ289091.1	NW_019168530.1	Pstpip2 (19201)
MG-4261	Gap	KQ030493.2	NW_012132909.2	Baalc (118452)
MG-3627	Gap	KZ289084.1	NW_019168523.1	Dnah12 (110083)
MG-3490	Clone Problem	KZ289082.1	NW_019168521.1	Ahnak2 (100041194)
MG-3618	Gap	KZ289070.1	NW_019168509.1	Efcab7 (230500)
MG-51	Gap	KZ289069.1	NW_019168508.1	Kazn (71529)
MG-89	Gap	KZ289066.1	NW_019168505.1	Intu (380614)
MG-74	Clone Problem	KZ289086.1	NW_019168525.1	Anxa13 (69787)
MG-209	Clone Problem	KZ289088.1	NW_019168527.1	Trerf1 (224829)

Table 1: Examples of issues fixed in GRCm38.p6 using assembled Illumina reads.

Figure 1 Top: Incomplete representation of Anxa13 gene in GRCm38 due to a deletion in reference component AC152395.9. Middle: clone error corrected in GRCm38.p6. Fix patch uses MF597750.1 and MF597749.1 to add deleted sequence to AC152395.9. It also provided a complete representation of Anxa13. Bottom: Representation of Anxa13 by reference chr. 15 and fix patch highlighting complete representation of Anxa13 (NM_027211.2).

GRCh38.p11: Clinically Relevant Updates to SLC39A4

noreply@blogger.com (grc) — Wed, 13 Sep 2017 11:44:00 +0000

The GRCh38.p11 patch release includes the fix patch scaffold KZ208914.1/NW_018654716.1 that updates two bases in SLC39A4, a change prompted by user request and that has implications for clinically-relevant variant analyses of this gene. This GRCh38 fix patch restores the gene to the same representation found in GRCh37.

SLC39A4, solute carrier family 39 member 4 (NCBI Gene ID: 55630), encodes a protein that is required for dietary zinc absorption in the intestine. In GRCh37, the BAC clone AF205589.5 (CTA-393G12) provided the sequence for SLC39A4 on chromosome 8. In GRCh38, AC233992.5 (RP11-735F20) replaced AC110280.8 (CTD-3232M19), the BAC clone upstream and adjacent to AF205589.5. As a consequence of this change, the default switch point in the overlap between these two clones led to the newly added RP11 clone providing the underlying sequence for SLC39A4 in GRCh38. A user subsequently contacted the GRC to report that this GRCh38 update introduced changes at two bases, a G to C substitution at CM000670.2/NC_000008.11: 144,414,297 (rs1871534) and a G to A substitution at CM000670.2/NC_000008.11: 144,415,811 (rs1871533). The “A” allele in GRCh38 has a global MAF of only 0.0098, according to data from the 1000 Genomes project, making it a rare allele. Additionally, the GRCh38 haplotype confounds clinical prediction algorithms at this locus because the p.Leu372Val (rs1871534) allele found in GRCh38 is incompatible with the p.Leu372Pro variant, which is implicated in Acrodermatitis entropathica (PMID: 12032886).

Review of Illumina genome sequencing data from an RP11 paired end WGS library (SRR834589) aligned to GRCh38 confirms the sequence of AC233992.5, the RP11 BAC clone providing the SLC39A4 sequence in GRCh38. Even though there are no sequence errors at these locations, the GRC agreed to further update these sites to address the needs of clinical researchers and to reduce the presence of globally rare alleles in the reference. Changing the switch point between the two GRCh38 BAC components allows the clone AF205589.5 to once again provide the sequence for SLC39A4 (Figure 1), and review by RefSeq annotators confirmed that this update addresses the two bases without introducing other changes that negatively affect gene representation. These updated bases are now available in the fix patch, and will be incorporated into the primary chromosome at the next major assembly release (GRCh39, currently unscheduled).

GRCh38.p11: Update to GCNT2

noreply@blogger.com (grc) — Tue, 11 Jul 2017 11:04:00 +0000

The GRC prioritizes curation efforts that improve gene representation in the human reference genome assembly. In some cases, such curation takes the form of base-pair level edits. The recent GRCh38.p11 patch release includes a new, curated, representation for the GCNT2 gene. The representation of the GCNT2 gene in the GRCh38 reference assembly contains the "C" allele for SNP rs539351 on chromosome 6 (NC_000006.12) at position 10,586,805, which reflects the sequence from the underlying component AL358777.12 (RP11-421M1) (Figure 1, top). During human development, the fetal blood group antigen (i) is converted to the adult antigen (I) by a beta-1,6-N-acetylglucosaminyltransferase-2 (GCNT2). Alternative splicing of the gene generates 3 isoforms, which differ only in their first exon. The SNP rs539351 is found in the first exon unique to the GCNT2 isoform C, which is the only one expressed in red blood cells, where this conversion occurs (NM_145655.3: c.816C>G (NP_663630.2: p.Asp272Glu). A user contacted the GRC with information that the reference allele had previously been described as a rare allele [1].

Although the reference assembly does not provide the most common alleles for all loci, the GRC does make an effort to make sure that reference alleles are not universally rare (defined for reference purposes as those with a global MAF < 5%), provided that it can do so while representing a biologically valid haplotype and a functional allele. Data from the 1000 Genomes project revealed that the "C" allele in GRCh38 had a global MAF=0.017. Thus, this allele was in scope for an update.

The GRC used sequence from ABBA01022081.1, a component of the HuRef assembly, as a new assembly component to provide the more common G allele at this position (Figure 1, bottom). We used haplotype information provided by Ensembl to confirm that the new coding representation is one that is biologically valid (GCNT2: 272D>E). This update is now included in the fix patch (KZ208911.1). This update should improve reviewing variation analyses results in which the reference assembly is being used as a model. The GRC continues to make these base updates for GRCh38. If you have questions or concerns about this process, let us know.

Figure 1 Top: Zoomed-in graphical view of the GCNT2 gene in GRCh38. The assembly sequence is shown at the top. The GCNT2 is shown in green. The reference allele D272 is a minor allele (brown box). Bottom: Zoomed-in graphical view of HG2057_PATCH, represents the more common allele (G) from ABBA01022081.1 (red box).

Reference:

Reid M., et al. The Blood Group Antigen FactsBook (3rd Edition), 603–608 (2012)

Improvements in the 5S rRNA gene cluster on chromosome 1q42.11-q42.13

noreply@blogger.com (grc) — Wed, 28 Jun 2017 11:17:00 +0000

Sequence updates that improve gene representation in the human reference genome assembly are priorities for the GRC. The recent GRCh38.p11 patch release includes a newly curated representation of the 5S RNA gene cluster (RN5S1@) located on chromosome 1q42.11-q42.13. The 5S ribosomal RNA (rRNA) is a component of the large subunit of the ribosome in all organisms. In humans, the 5S rRNA cluster is comprised of individual rRNA genes repeated in head-to-tail orientation with non-rRNA sequences in the spacer regions. The number of 5S rRNA repeats per haploid human genome is highly polymorphic, in a range of 35-175 (1).

The repetitive clustered nature of the 5S rRNA region has long complicated both its sequencing and assembly, and its representation is incomplete in GRCh37 and GRCh38, the last two major reference assembly versions, though in different ways. The underlying components AL139288.15 (RP5-915N17) and AL713899.14 (RP4-621O15) provide the sequence for the 5S rRNA region in both assemblies. In GRCh37, a false alignment between repeat copies in the two components led to a contiguous, but collapsed, representation. In GRCh38, the false alignment was broken and a default 50 kb gap was inserted in chromosome 1 (CM000663.2/NC_000001.11) at 228,558,365 bp as a placeholder for the missing sequence (Figure 1, top). The GRCh38 representation of the cluster includes only 19 5S rRNA gene unit copies (17 functional and 2 pseudogenes) (Figure 1, top).

The fix patch (KZ208906.1) included in the GRCh38.p11 release now provides a contiguous and validated representation of the 5S rRNA genomic region. The patch closes the assembly gap and replaces the 5S rRNA copies from AL139288.15 and AL713899.14 with sequences from AC275639.1 (CH17-275P10), a BAC clone that completely spans the cluster (Figure 1, bottom). The patch provides 35 copies (34 functional and a single pseudogene) of 5S rRNA genes (Figure 1, bottom). The haplotype represented in this clone has been verified by BioNano optical map data for the haploid CHM1 sample, from which the clone library was derived. This new representation should serve as an improved substrate for analysis of the region, including read alignment and variation analysis.

Figure 1 Top: 5S rRNA region in GRCh38. Incomplete representation of 5S rRNA gene cluster in GRCh38 due to an assembly gap. Bottom: 5S rRNA fix patch in GRCh38.p11. The gap is closed and a complete representation of the 5S rRNA is provided.

Reference:

Stults, DM. et al. Genome Res. 18(1):13-8 (2008)

GRCz11 – the latest zebrafish reference genome assembly

noreply@blogger.com (grc) — Tue, 23 May 2017 08:45:00 +0000

After 2.5 years of assembly curation, the GRC is proud to present the new zebrafish reference genome assembly, GRCz11.

This latest assembly has been refined by the addition of nearly 1000 finished clone sequences and by the resolution of more than 400 assembly issues. This resulted in a significant reduction in scaffold numbers (3399 to 1905) and increase in scaffold N50 (2.18 Mb to 7.5 Mb) whilst the overall genome size was not affected. Figure 1 shows an overview of contig and scaffold N50s over time, indicating the advance in assembly curation.

Figure 1: Contig vs. scaffold N50s for zebrafish reference genome assemblies. Release dates: Zv7: 2008, Zv8: 2009, Zv9: 2010, GRCz10: 2014, GRCz11: 2017.

Alignments of 16133 RefSeq sequences showed a further improvement over past assemblies: only 31 sequences remained not found (down from 34), 105 transcripts are still split between locations (down from 205) and only 441 exhibit less than 95% CDS coverage (down from 566). Figure 2 shows an example of an improved region, correcting the representation of two genes.

Figure 2: gEVAL screenshot of the supt4h1 gene (red arrow) in GRCz10 (top) and GRCz11 (bottom). In GRCz10 the supt4h1 gene on chromosome 5 is incomplete, missing its first exon, and surrounded by a truncated supplicated copy of rnf150b (blue arrow). In GRCz11, the supt4h1 gene is complete and neighbouring the hsf5 gene, as seen in other vertebrates, whereas the rnf150b gene is now complete and located singularly on chromosome 23. gEVAL, the GRC’s genome assembly evaluation browser, indicates completeness of genes and other features via colours (green > 98% coverage, yellow = 50-98% coverage, red < 50% coverage).

GRCz11 was built as described previously using clone sequences ordered and oriented according to genetic markers and BioNano data, the latter greatly influencing the scaffolding. Remaining gaps were filled with selected contigs from whole genome sequencing assemblies, mainly WGS31, and in a few cases, WGS32.

For the first time in a zebrafish assembly, GRCz11 also features alternate loci scaffolds (ALT_REF_LOCI). The alternate loci represent variant sequence representations for certain genomic regions. They were selected from a pool of 1895 finished clones that were found to inhabit an assembly region already occupied by clone sequence and were therefore not included in the primary chromosomal path. All surplus clones that exhibited at least 5 kb of unique sequence not present in the primary chromosomal path were added to the assembly as alternate loci scaffolds, totaling 186 Mb of additional sequence in 1150 clones. The alignments of the alternate loci scaffolds to the primary chromosomal path are also included in the GRCz11 assembly to provide the chromosome context for these alternate sequences. The alternate loci will be represented in genome browsers in the same way as human and mouse ALT_REF_LOCI. Additional smaller scale variation will be submitted to dbSNP/dbVAR/EVA.

After release of this assembly, within the GRC, the Sanger team is transferring the maintenance of the zebrafish reference to the new GRC member ZFIN. ZFIN will take on the future curation of the assembly, and invites user reports on assembly issues. Future updates to the assembly will be issued as patch releases, adding sequence but not impacting the chromosomal coordinates.

New haplotype representations in the LRC_KIR region on chromosome 19q13.4

noreply@blogger.com (grc) — Tue, 08 Nov 2016 13:04:00 +0000

The KIR (Killer cell immunoglobulin-like receptor) gene cluster is a region of approximately 150 kb within the Leukocyte Receptor Complex (LRC) on human chromosome 19q13.4 (CM000681.2: 50900001-58617616). The KIR family is highly divergent, with multiple haplotypes differing in gene content, and with individual genes exhibiting allelic variation. Only a few KIR genes are conserved between humans and chimpanzees, the closest living relatives to humans. The GRCh38 assembly includes 35 representations for the LRC-KIR region as alternate loci scaffolds in addition to the chromosomal sequence (ALT_REF_LOCI in LRC-KIR region). David Roe and colleagues (personal communication) recently sequenced a diverse panel of KIR haplotypes with the goal to report novel structures and to demonstrate the ability to fully characterize KIR haplotypes, even across long repetitive sequences in diploid individuals.The sequences for these 15 haplotypes are derived from 8 individuals (one is homozygous for the haplotype). While all 15 haplotypes represent new allelic variations, 6 of the 15 haplotype structures exhibit novel numbers and types of KIR genes (rows highlighted black in Table 1).

KIR_haplotype	GenBank_ID	RefSeq_ID
cA01-tA01	KV575246.1	NW_016107300.1
cA01-tA01	KV575247.1	NW_016107301.1
cA01-tA01	KV575248.1	NW_016107302.1
cA01-tB04	KV575249.1	NW_016107303.1
cA01-tB01	KV575250.1	NW_016107304.1
cA01-tA01	KV575251.1	NW_016107305.1
cB05-tA01	KV575252.1	NW_016107306.1
cA01-tA01	KV575253.1	NW_016107307.1
cA03-tB02	KV575254.1	NW_016107308.1
cA01-tA01	KV575255.1	NW_016107309.1
cB01-tB01	KV575256.1	NW_016107310.1
cA04	KV575257.1	NW_016107311.1
cB04-tB03	KV575258.1	NW_016107312.1
cA01-tA01	KV575259.1	NW_016107313.1
cB02-tA01	KV575260.1	NW_016107314.1

Table 1: Haplotype identifier, GenBank and RefSeq accession numbers for the 15 new KIR haplotypes reported in GRCh38.p8; rows highlighted in black indicate new haplotype structures. The haplotype name shown in Table 1 is reported in the Comment section in both GenBank and RefSeq records.

All 15 new haplotypes have been added to the reference assembly in GRCh38 patch release 8 (GRCh38.p8) as novel patches. Figure 1 provides a comparison of the KIR gene content of reference chr. 19 and the novel patch haplotype "cA04". The novel patch NW_016107311.1 represents a new KIR haplotype which contains only 4 KIR genes as compared to 9 KIR genes in the reference chr. 19.

Figure 1: LRC_KIR region on chromosome 19q13.4. Top: KIR region on reference chromosome 19, the 4 genes in common between the reference chromosome and the novel patch labeled in red and the 5 genes missing in haplotype "cA04" boxed in blue. Bottom: KIR gene represntation in haplotype "cA04" (KU645198.1). This haplotype has only 4 KIR gene (labeled in red) as compared to the reference chr. 19. The blue bars represent the tiling path of chr. 19 (NC_000019.10) and the gray bar represents novel sequences. In the gray track representing alignment of KIR haplotype to chr. 19, the solid horizontal red line indicates the region of KIR haplotype "cA04" that has no alignments to reference chromosome, the small vertical red bars indicate mismatches, and site of missing sequence in “cA04” is highlighted as deletion.

The Roe group assisted the GRC with the alignments of the patch scaffolds to the reference chromosome to ensure gene relationships were properly represented. The KIR region is one of the most variable regions of the human genome. Donor selection based on KIR genotyping of multiple best matched HLA has improved transplantation outcome, and certain combinations of KIR-HLA genotypes have been reported in association with autoimmune diseases and malignancies. The newly represented KIR haplotypes provide additional resources for haplotype analysis of KIR genes.

Are you familiar with CYP2D6 and its importance in drug metabolism?

noreply@blogger.com (grc) — Mon, 24 Oct 2016 19:42:00 +0000

CYP2D6 (Gene ID: 1565) is a gene associated with the metabolism of ~25% of clinically prescribed drugs, including antidepressants, neuroleptics and opioids. CYP2D6 is located on chromosome 22 (22q13.1), near two cytochrome P450 pseudogenes CYP2D7 and CYP2D8P. Because of its functional importance, the GRC updated the chromosomal representation for CYP2D6 to the sequence-corrected clinical standard (CYP2D6*1A) in GRCh38. The version of CYP2D6 found in the prior assembly version, GRCh37, was retained in GRCh38 as an alternate loci scaffold (KI270928.1).

The Genome Reference Consortium, in conjunction with the Pharmacogenomics Research Network, has also sought to identify and provide reference assembly representation for structural variation at the CYP2D6 locus. Much of this work was done by examining the end-sequence alignments of different fosmid libraries to the reference (Kidd et al.). As the reference assembly was known to represent CYP2D6, CYP2D7 and CYP2D8P each in single copy, we could ascertain potential duplication and deletion alleles of these genes by identification of discordant fosmid end-sequences (Figure 1).

Figure 1. Alignment of fosmid ends from the ABC12 library to GRCh38 chr. 22 in the vicinity of CYP2D6. Lines connect ends belonging to the same clone. Concordant placements (length within 3 standard deviations of the library insert average and inward facing ends) are shown in blue; clones with discordant placements are in red.

As of the GRCh38.p7 assembly release, there are 3 alt loci and 3 novel patch scaffolds that provide representation for significant structural variation in the CYP2D6 region (Table 1). Figure 2 shows the alignment of these scaffolds to the reference chromosome, highlighting the diversity in the variant representations. An example of a CYP2D6 triplication haplotype is shown in Figure 3.

Table 1: Scaffolds providing alternate sequence representations for the CYP2D6 region, as of GRCh38.p7.

The inclusion of these additional representations for the locus in the reference assembly is intended to help in the evaluation of CYP2D6 variant alleles from other samples. The variant scaffolds can be included in the target assembly when using an alternate aware aligner, such as bwa-mem or SRPRISM, to align reads and should enable the identification of the haplotype that is the closest match to the query sample.

Figure 2. Alignment of alt loci and patch scaffolds to the CYP2D6 region of GRCh38 chr. 22. The blue bars at top represent the genomic clones comprising chr. 22. The NCBI RefSeq annotation of the chromosome is shown in the middle. The grey bars at the bottom are the alignments. Thin horizontal red lines represent deletions in the scaffolds relative to the chromosome, while vertical blue bars represent the locations of scaffold insertions.

Figure 3. Graphical view of patch scaffold NW_014040931.1, with the alignment of CYP2D6, revealing a haplotype containing a triplication of this locus.

ZFIN joins the GRC

noreply@blogger.com (grc) — Tue, 17 May 2016 18:17:00 +0000

ZFIN, the zebrafish model organism database, is joining the GRC and is planning to take over the maintenance and curation of the zebrafish genome assembly. ZFIN curators visited with GRC curators at the Wellcome Trust Sanger Institute in January 2016 to undergo training in the use of GRC tools and processes. ZFIN anticipates taking over responsibility after the next planned release of the zebrafish assembly, GRCz11. A specific timeline for this release has not yet been established, but is likely to be towards the end of 2016. ZFIN curators will continue training through 2016 with GRC curators on zebrafish genome issues.

ZFIN will respond to potential problems in the assembly that are identified by researchers. Users can report issues on the assembly via the Report an Issue link at the GRC website. There are currently no plans to actively identity problems in the assembly. Once problems are brought to the attention of ZFIN GRC curators, updates will be issued as patches.

The GRC welcomes your feedback on this update.

Chicken assembly curation at GRC

noreply@blogger.com (grc) — Mon, 18 Apr 2016 14:52:00 +0000

Upon the release of Gallus_gallus-5.0 (GCA_000002315.3), the GRC assumed responsibility for the continued curation of the chicken reference genome assembly from the International Chicken Genome Consortium. The assembly represents the Red Jungle Fowl strain, inbred line UCD001. All sequences in the assembly are derived from a single individual from this line, female "RJF #256". The assembly is a hybrid comprised primarily of WGS contigs, into which genomic clones have been integrated. Planned curation efforts are focused on selecting genomic clones (BACs) for sequencing and assembly, specifically from the CHORI-261 library, to fill known gaps and resolve assembly errors.

Figure 1. Ideogram of Gallus_gallus-5.0 (GCA_000002315.3)

Information about the current assembly and curation efforts is now available on the GRC website. An interactive interface (Figure 2) provides access to genome regions currently under review, while a series of issue pages provide details and mapping information for each of these regions, as well as a graphical view (Figure 3). For more information on these pages, see our previous blog posts on issue pages and the issue overview page.

Figure 2: Overview of issues reported on the chicken reference genome assembly.

Figure 3: Example of page with issue-specific details

The GRC welcomes feedback on the current assembly from members of the chicken research community. Users can either Report an Issue or Contact Us for more information about the assembly. A survey regarding the usability of the GRC website is also ongoing.

Updates to DUX4 region on subtelomeric chromosome 4q

noreply@blogger.com (grc) — Thu, 07 Apr 2016 11:26:00 +0000

Sequence improvements and increased variant representation in the human genome reference assembly are priorities for the GRC. This blog post describes two updates in the recent GRCh38.p7 patch release affecting the DUX4 region located at the chromosome 4q sub-telomere: (1) a FIX patch correcting the chromosomal representation (KQ983257.1) and (2) a NOVEL patch representing a variant of the region (KQ983258.1). A third haplotype of DUX4 region which is represented in GRCh37 will be also described.

The DUX4 region contains tandem arrays of a 3.3 Kb D4Z4 macrosatellite repeat located in the sub-telomeric region of chromosome 4q. The number of D4Z4 repeats is highly polymorphic, ranging from 8 to 100 copies in the healthy individuals, but only 1 to 10 units in individuals with Facioscapulohumeral muscular dystrophy-1 (FSHD1: MIM#158900). The contraction of the repeat arrays is believed to result in a decreased epigenetic repression effect of D4Z4 and subsequent transcriptional activation of the DUX4 gene. Three main haplotypes, known as 4qA, 4qB and 4qA-L, have been reported for this region (1-3). The 4qB haplotype has homology to 4qA in the D4Z4 repeats, but is completely different in the distal region (1,2). 4qA, considered the reference haplotype, is the ancestral and most common haplotype. 4qA and 4qA-L are associated with FSHD, while 4qB is not (2). The different haplotypes exhibit population stratification (3).

The GRC received user feedback that the DUX4 region is incorrectly represented on chromosome 4 of the GRCh38 assembly. The repeat structure in the region complicates both sequencing and assembly. Clones representing both the 4qA and 4qB haplotypes are present in GRCh38, creating a haplotype expansion and accompanying "false" gap at NC_000004.12: 190,123,122 bp, between components AC225782.3 (WI2-3035O22; 4qB) and AC215524.3 (ABC7-42391500H16; 4qA (partial)) (Figure 1, top). In the GRCh38.p7 fix patch scaffold KQ983257.1, the GRC provides a complete representation for the 4qA haplotype by replacing AC225782.3 with CT476828.7 (RP11-242C23) and eliminating the gap (Figure 1, bottom). The patch provides 14 copies of the DUX4 repeat. This fix patch representation for the region will be incorporated into chromosome 4 in the as yet unscheduled GRCh39 assembly release, and the 4qB representation currently present on the GRCh38 chromosome will be provided as an alternate loci scaffold.

Figure 1 Top: DUX4 region in GRCh38. Incomplete representation of DUX4 gene in GRCh38 due to a mix haplotype representation of 4qB and partial 4qA. Bottom: DUX4 fix patch in GRCh38.p7. The gap is closed and a complete representation of the 4qA haplotype is provided.

The GRCh38.p7 release also includes KQ983258.1 as novel patch scaffold providing representation for the variant 4qA-L haplotype. This variant is very similar to haplotype 4qA (Figure 2, top, green dashed line) but diverge in the distal-most DUX4 copy (Figure 2, top, red dashed line) which is about 1.6 Kb larger in 4qA-L (Figure 2, bottom). The functional implication of this difference is not yet known.

Figure 2 Top: Novel patch representing DUX4 variant 4qA-L. This variant is similar to haplotype 4qA but the region distal to last D4Z4 unit is longer in 4qA-L. Bottom: Schematic of D4Z4 repeat arrays for 4qA and 4qA-L regions, adapted from Lemmers et al., 2010.

References:

1. van Geel, M. et al. Genomics 79, 210–217 (2002)

2. Lemmers, RJLF. et al. Nature Genetics 32, 235-236 (2002).

3. Lemmers, RJLF. et al. Science 329, 1650-1653 (2010).

GRC reference assembly curation with BioNano maps

noreply@blogger.com (grc) — Wed, 09 Mar 2016 14:39:00 +0000

Commercial whole genome mapping systems, such as OpGen and BioNano, are playing an increasingly important role in a variety of genomic analyses, including de novo assembly, structural variant detection and assembly curation.

Comparison of a reference assembly to a collection of whole genome maps can help curators find potential regions of misassembly and identify genomic variations that are candidates for representation in alternate loci scaffolds. For example, optical maps played a key role in the GRC's resolution of the human 10q11.22 tiling path, as described in this prior GRC blog post.

To assist with curation efforts, the Wellcome Trust Sanger Institute, a GRC member, has integrated BioNano data sets for human, mouse and zebrafish assemblies into the gEVAL browser. They have also provided data from OpGen maps and optical maps from David Schwartz (PMID: 20534489). Common curation use cases for these browser data include:

Gap sizing
Confirmation of assembly components
Identification of problems in assembly components
Identification of assembly regions missing sequence
Detection of haplotype over-expansions

Below is an example highlighting the potential usage.

The issue (HG-172) reports possible assembly error or missing sequence from reference component AL691432.54 that affects CDC2L1 (GeneID:984) in GRCh37. Navigating to this region in gEVAL and turning on the tracks for:

REFSEQ transcript mappings
BspQI insilico digest
JIRA issue entries
Bionano genome maps

NA12878
NA24143 (Ashkenazim trio mother)
NA24149 (Ashkenazim trio father)
NA24385 (Ashkenazim trio son)
NA24631 (Chinese trio son)

In the REFSEQ track there are 4 distinct transcript mapping groups, in which most are orange colour indicating the mapping is incomplete in terms of coverage. This indicates that perhaps there may be an issue in the assembly. From left to right the first group is mib2, mm23A/B, cdc2L1(cdk11b) and slc35e2.


Jump to this Region in gEVAL

Zooming into the region of the red features in the Bionano genome maps reveals clearly the fragment sizes between Bionano BspQI nicks/labels. The red features within each map indicates a discordance in size with the BspQI insilico digest.

The insilico digest reveals that the region in question on the assembly has a fragment size of ~26,5kb, whereas all 5 genome maps reveal two fragments totaling ~30kb. This discordant region encompasses the cdc2L1 gene (which from further analysis indicates 2 exons missing) as well, giving evidence that perhaps this region is not represented correctly and missing roughly 3-4kb of sequence.

Upon further review and analysis, the GRC curation team was able to place in to motion new clone sequence to represent correctly this region. The new path is represented in GRCh38 below. The addition of clone FO704657 on to the assembly path correctly contains the complete coding region for the cdc2L1(cdk11b) gene. It also corrects the flanking gene mappings as well. This is indicated by the green mappings of the REFSEQ transcripts. The discordant red features of the Bionano genome maps are no long present, replaced with concordant fragment mappings. The issue can be updated as resolved.

Jump to this Region in GRCh38

For more information on how to use BioNano displays in gEVAL, check out the gEVAL browser blog.

GRC Website: Individual Genome Issues

noreply@blogger.com (grc) — Thu, 19 Feb 2015 15:41:00 +0000

Are you looking for the latest status updates from the GRC on the human, mouse or zebrafish reference genome assemblies? In companion to our previous post, we now explain how to use the Individual Genome Issue reports on the GRC website. As described in our last blog, you can filter and search for issues of interest using the organism-specific "Issues Under Review" pages. To provide an example for this blog post, we applied the follow filtering options on the human "Issues Under Review" page: issues location = GRCh38.p2, chromosome = chr3 , type = variation and scaffold type = ALT (alternative loci). We then selected HG-1291 from column 1 of the results table to go to the individual issue page, shown below in Figure 1. On this and other individual issue pages, you'll find the following information:

Summary fields describing the issue and its latest status updates or resolution (blue box)
Ideogram showing the issue's genomic location (green box)
Patch and/or alternate loci status and history (orange box)
Graphical view of genomic region to which issue is mapped (red box).

Note: graphical views are provided for all mapped locations in the previous and current assembly versions. For example, HG-1291 has been mapped to chr. 3 and an alternate locus scaffold in GRCh38.p2, and to chr. 3 and a novel patch in GRCh37.p13. Use the radio buttons to toggle the display between the different sequence locations.

Figure 1. GRC Issue page for HG-1291, with page features highlighted.

Below, figure 2 shows the graphical view of the GRCh38.p2 alternate locus scaffold to which HG-1291 has been mapped (NW_003871060.2). Default tracks in the graphical views provide you with additional information about the assembly composition and quality. They include:

Assembly components
Alignments of alternate loci/patch scaffolds to the primary assembly
Annotated component assembly problems
All GRC issues mapped to the region
NCBI Gene annotation
Ensembl Gene annotation

In this image of HG-1291, review of the Genes and Alignment tracks reveals two exons in a region of the alternate loci that has no alignment to the chromosome (arrow and circle). This annotation supports the description in the Issue Summary fields. You can further configure the tracks or upload your own data files to the graphical view by clicking on the "Configure" button at the top right of the viewer (red box).

Figure 2. Graphical view of NW_003871060.2, the GRCh38.p2 alternate loci scaffold to which issue HG-1291 is mapped. The exons captured by the additional sequence in the scaffold are highlighted.

If you have questions about any of the issues you see, please contact the GRC and reference the issue number. If you know of a genome issue that isn't found on these pages, please report the issue to the GRC.

GRC Website Update: Genome Issues Under Review

noreply@blogger.com (grc) — Tue, 03 Feb 2015 16:35:00 +0000

GRC "Genome Issues under Review" webpage update!

Do you know how to find genome issues on the GRC website? To get started, select an organism from the top of the GRC homepage, and in the corresponding organism overview page select the link for "Issues Under Review". These pages provide you with the latest information about potential problems and other issues related to the human, mouse and zebrafish reference genome assemblies that the GRC are working on. Recent updates to these pages make them more interactive, informative and easier to navigate so you can pinpoint issues relevant to your research interests. Some of the page features are highlighted in Figure 1, which shows "Human Genome Issues".

Show issue locations on (blue box): Use this to define the assembly version on which you want to see mapped issues. We support issue mapping to the current assembly and the last release of the prior assembly version.

Ideogram (green box): The histogram above presents the number of issues related to each chromosome, and the annotations show issue locations. Looking for issues related to a single chromosome? Click on a chromosome or histogram of interest to see a more detailed ideogram with annotated issues (more on this below).

Search (purple box): Use this to finding issues related to a specific gene/clone/accession number/chromosomal location.

Data table: Provides a summary of issues. Within this table, click on issue ID (brown box) to go to web pages for specific issues or View in browsers (brown box) to see the relevant genome regions in browsers at Ensembl, NCBI, and UCSC.

Figure 1. Human Genome Issues overview

Additional page features shown below in Figure 2 will help you identify the issues that interest you most:

Filter: Located to the left of the data table, this section contains various display filters, including issue type and issue status, to help you find GRC issues meeting specified criteria.
Issue Annotations: In the single chromosome ideogram displays, issues are annotated below the figure.

Tool-tips: Click on any annotation for a summary and a link to the issue page
Bar charts: Click on either of the interactive bar charts below the ideogram to re-categorize the issue annotation display by Type or Status.

Figure 2. Chromosome 1 genome issues

GRCh38: Patching the ABO gene

noreply@blogger.com (grc) — Wed, 28 Jan 2015 14:53:00 +0000

GRCh38 has started receiving patch updates, and this blog post describes a FIX patch to the ABO gene, located on chr. 9. You might have been aware that the GRC released a FIX patch to ABO for GRCh37. So why is there an ABO FIX patch for GRCh38 as well?

In GRCh37, the ABO gene was annotated on sequence derived from two RP11 library clones, AL732364.9 (RP11-244N20) and AL158826.23 (RP11-430N14). However, the RP11 library is derived from a diploid genome and analysis demonstrated that the two sequenced clones represented two different Type O ABO alleles. As a result, the GRCh37 chr.9 representation of ABO was an invalid haplotype for the gene (Fig. 1, top panel).

Fig. 1 Top: ABO region in GRCh37. The gene is derived from 2 components, resulting in an invalid haplotype not seen in any individuals. Bottom: ABO fix patch. The gene is derived from a single component and represents a known Type O haplotype.

To address this issue, we identified a clone from the CalTech human BAC library D that captures the complete ABO gene (CTD-2612A24). The sequence for this clone was finished (AL772161.10) and inserted into the chr. 9 tiling path, replacing RP11 component AL158826.23. By setting the switch points between AL732364.9 (RP11-244N20) and AL772161.10 (CTD-2612A24) so that the full insert sequence of the new component contributed to the scaffold, we were able to provide a complete and valid ABO Type A1.02 representation for the gene. Thus update was provided as a FIX patch scaffold (GL339450.1) for GRCh37. (Fig.1, bottom panel).

Unfortunately, this update is not reflected in GRCh38. Subsequent to the final GRCh37 patch release (GRCh37.p13) and the release of GRCh38, the sequence to RP11-244N20 was updated (AL732364.10) and inserted into the chr. 9 tiling path. The switch points between the updated sequence AL732364.10 and AL772161.10 were set incorrectly (Fig. 2). This resulted in an invalid haplotypic representation for ABO. Whereas the GRCh37 representation was a Type O/O mix, in GRCh38 it is a Type A/O mix.

Fig.2 Top: ABO fix patch. Gene is derived from a single component. Bottom: ABO region in GRCh38. The gene is derived from 2 components, creating an invalid haplotype. This is fixed by the GRCh38 FIX patch.

The GRCh38 FIX patch scaffold KN196479.1 corrects this switch point and provides the same single haplotype representation for ABO that was present in the GRCh37 FIX patch scaffold. This re-patching of the ABO gene again restores the functionality of the gene with the valid Type A1.02 haplotype.

Optical Mapping data in the GRC track hub

noreply@blogger.com (grc) — Thu, 27 Nov 2014 15:31:00 +0000

The GRC track hub now includes Optical Mapping analysis information.

What is Optical Mapping?
Optical Mapping (OM) is a method to produce ordered restriction maps from single DNA molecules (rMaps). These rMaps are assembled into consensus maps which can be aligned against the reference assembly, taking into account the positioning of restriction sites and length of fragments. OM aids the scaffolding of genomic sequence and the identification of errors in genome assemblies, but it is also very helpful in confirming assembled contigs and sizing gaps.

What OM data is available?
OM data is currently available for human (Teague et al., 2010) and mouse (Church et al., 2009) and we would like to thank Steve Goldstein and David Schwartz for providing the alignments to the respective reference assemblies.

What is displayed in the GRC track hub?
The OM data is divided into several tracks in the GRC track hub. These tracks are of three types:

OM alignment tracks show the alignments of consensus maps to the reference genome, based on the comparison of restriction patterns. Each track of this type corresponds to an analysis of OM data from a single cell line.
OM deletion tracks present the locations of additional restriction fragments that have no corresponding fragment in the reference assembly. Their position is defined by the remaining alignment of the respective consensus map. Again, each track of this type corresponds to an analysis of OM data from a single cell line.
Each assembly also has a single OM reference track, which presents the set of OM fragments that would be expected based on the reference sequence, produced via an in silico restriction digest.

How is this information visualised?
The way that OM analysis data is displayed is slightly different for each of the types of track mentioned above (alignments, deletions, and predicted fragments based on the reference).

The OM alignments tracks present each contig as a horizontal line, with restriction cut-sites dividing fragments being displayed as vertical lines along that contig. Where there is a space between the placement of successive restriction fragments according to this analysis, this is represented as a thicker vertical bar spanning the gap between the fragments.
The OM deletions tracks use a single vertical bar to show the location of each fragment or group of fragments with no corresponding fragment in the reference assembly. The size of the fragment is not represented by the glyph in the browser, but is shown as one of its data fields.
The OM reference track displays the cut-sites between expected restriction digest fragments as vertical lines.

Display examples
In the Ensembl genome browser (from version 78 onwards), the OM reference track is at the top, with the OM deletions track "OM gap 15510" below it, followed by three OM alignments tracks based on different cell lines. (Note that OM deletions tracks exist for all those cell lines which have alignments tracks, but only one OM deletion track has data at this location.)

Here is how it appears in the UCSC genome browser. The tracks are in the same order as for the Ensembl example above: the OM reference track is at the top, with the OM deletions track "OM gap 15510" below it, followed by three OM alignments tracks based on different cell lines.