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June 1, 2021  |  

HLA variant identification techniques

The Human Leukocyte Antigen (HLA) genes located on chromosome 6 are responsible for regulating immune function via antigen presentation and are one of the determining factors for stem cell and organ transplantation compatibility. Additionally various alleles within this region have been implicated in autoimmune disorders, cancer, vaccine response and both non-infectious and infectious disease risk. The HLA region is highly variable; containing repetitive regions; and co-dominantly expressed genes. This complicates short read mapping and means that assessing the effect of variation within a gene requires full phase information to resolve haplotypes.One solution to the problem of HLA identification is the use of statistical inference to suggest the most likely diploid alleles given the genotypes observed. The assumption of this approach is the availability of an extensive reference panel. Whilst there exists good population genetics data for imputing European populations, there remains a paucity of information about variation in African populations. Filling this gap is one of the aims of the Genome Diversity in Africa Project and as a first step we are performing a pilot study to identify the optimal method for determining HLA type information for large numbers of samples from African populations.To that end we have obtained samples from 125 consented African participants selected from 5 populations across Africa (Morrocan, Ashanti, Igbo, Kalenjin, and Zulu). The methods included in our pilot study are Sanger sequencing (ABI), NGS on HiSeqX Ten platform (Illumina); long-range PCR combined with single molecule real-time (SMRT) sequencing (PacBio); and for a subset of samples library preparation on GemCode Platform (10x Genomics), which delivers valuable long range contextual information, combined with Illumina NGS sequencing.Results from capillary sequencing suggests the presence of a minimum of two novel alleles. Long Range PCR have been performed initially on a subset of samples using both primers sourced from GenDX and designed as described in Shiina et al (2012). Initial results from both primer sets were promising on Promega DNA test samples but only the GenDX primers proved effective on the African samples, producing consistently PCR products of the expected size in the Igbo, Ashanti, Morrocan and Zulu samples. We will present early results from our evaluation of the different sequencing technologies

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June 1, 2021  |  

Building a platinum human genome assembly from single haplotype human genomes generated from long molecule sequencing

The human reference sequence has provided a foundation for studies of genome structure, human variation, evolutionary biology, and disease. At the time the reference was originally completed there were some loci recalcitrant to closure; however, the degree to which structural variation and diversity affected our ability to produce a representative genome sequence at these loci was still unknown. Many of these regions in the genome are associated with large, repetitive sequences and exhibit complex allelic diversity such producing a single, haploid representation is not possible. To overcome this challenge, we have sequenced DNA from two hydatidiform moles (CHM1 and CHM13), which are essentially haploid. CHM13 was sequenced with the latest PacBio technology (P6-C5) to 52X genome coverage and assembled using Daligner and Falcon v0.2 (GCA_000983455.1, CHM13_1.1). Compared to the first mole (CHM1) PacBio assembly (GCA_001007805.1, 54X) contig N50 of 4.5Mb, the contig N50 of CHM13_1.1 is almost 13Mb, and there is a 13-fold reduction in the number of contigs. This demonstrates the improved contiguity of sequence generated with the new chemistry. We annotated 50,188 RefSeq transcripts of which only 0.63% were split transcripts, and the repetitive and segmental duplication content was within the expected range. These data all indicate an extremely high quality assembly. Additionally, we sequenced CHM13 DNA using Illumina SBS technology to 60X coverage, aligned these reads to the GRCh37, GRCh38, and CHM13_1.1 assemblies and performed variant calling using the SpeedSeq pipeline. The number of single nucleotide variants (SNV) and indels was comparable between GRCh37 and GRCh38. Regions that showed increased SNV density in GRCh38 compared to GRCh37 could be attributed to the addition of centromeric alpha satellite sequence to the reference assembly. Alternatively, regions of decreased SNV density in GRCh38 were concentrated in regions that were improved from BAC based sequencing of CHM1 such as 1p12 and 1q21 containing the SRGAP2 gene family. The alignment of PacBio reads to GRCh37 and GRCh38 assemblies allowed us to resolve complex loci such as the MHC region where the best alignment was to the DBB (A2-B57-DR7) haplotype. Finally, we will discuss how combining the two high quality mole assemblies can be used for benchmarking and novel bioinformatics tool development.

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June 1, 2021  |  

Highly accurate read mapping of third generation sequencing reads for improved structural variation analysis

Characterizing genomic structural variations (SV) is vital for understanding how genomes evolve. Furthermore, SVs are known for playing a role in a wide range of diseases including cancer, autism, and schizophrenia. Nevertheless, due to their complexity they remain harder to detect and less understood than single nucleotide variations. Recently, third-generation sequencing has proven to be an invaluable tool for detecting SVs. The markedly higher read length not only allows single reads to span a SV, it also enables reliable mapping to repetitive regions of the genome. These regions often contain SVs and are inaccessible to short-read mapping. However, current sequencing technologies like PacBio show a raw read error rate of 10% or more consisting mostly of insertions and deletions. Especially in repetitive regions the high error rate causes current mapping methods to fail finding exact borders for SVs, to split up large deletions and insertions into several small ones, or in some cases, like inversions, to fail reporting them at all. Furthermore, for complex SVs it is not possible to find one end-to-end alignment for a given read. The decision of when to split a read into two or more separate alignments without knowledge of the underlying SV poses an even bigger challenge to current read mappers. Here we present NextGenMap-LR for long single molecule PacBio reads which addresses these issues. NextGenMap-LR uses a fast k-mer search to quickly find anchor regions between parts of a read and the reference and evaluates them using a vectorized implementation of the Smith-Waterman (SW) algorithm. The resulting high-quality anchors are then used to determine whether a read spans an SV and has to be split or can be aligned contiguously. Finally, NextGenMap-LR uses a banded SW algorithm to compute the final alignment(s). In this last step, to account for both the sequencing error and real genomic variations, we employ a non-affine gap model that penalizes gap extensions for longer gaps less than for shorter ones. Based on simulated as well as verified human breast cancer SV data we show how our approach significantly improves mapping of long reads around SVs. The non-affine gap model is especially effective at more precisely identifying the position of the breakpoint, and the enhanced scoring scheme enables subsequent variation callers to identify SVs that would have been missed otherwise.

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June 1, 2021  |  

Detection of structural variants using third generation sequencing

Structural Variants (SVs), which include deletions, insertions, duplications, inversions and chromosomal rearrangements, have been shown to effect organism phenotypes, including changing gene expression, increasing disease risk, and playing an important role in cancer development. Still it remains challenging to detect all types of SVs from high throughput sequencing data and it is even harder to detect more complex SVs such as a duplication nested within an inversion. To overcome these challenges we developed algorithms for SV analysis using longer third generation sequencing reads. The increased read lengths allow us to span more complex SVs and accurately assess SVs in repetitive regions, two of the major limitations when using short Illumina data. Our enhanced open-source analysis method Sniffles accurately detects structural variants based on split read mapping and assessment of the alignments. Sniffles uses a self-balancing interval tree in combination with a plane sweep algorithm to manage and assess the identified SVs. Central to its high accuracy is its advanced scoring model that can distinguish erroneous alignments from true breakpoints flanking SVs. In experiments with simulated and real genomes (e.g human breast cancer), we find that Sniffles outperforms all other SV analysis approaches in both the sensitivity of finding events as well as the specificity of those events. Sniffles is available at: https://github.com/fritzsedlazeck/Sniffles

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June 1, 2021  |  

High-quality, highly contiguous re-assembly of the pig genome

Many applications of high throughput sequencing rely on the availability of an accurate reference genome. Errors in the reference genome assembly increase the number of false-positives in downstream analyses. Recently, we have shown that over 33% of the current pig reference genome, Sscrofa10.2, is either misassembled or otherwise unreliable for genomic analyses. Additionally, ~10% of the bases in the assembly are Ns in gaps of an arbitrary size. Thousands of highly fragmented contigs remain unplaced and many genes are known to be missing from the assembly. Here we present a new assembly of the pig genome, Sscrofa11, assembled using 65X PacBio sequencing from T.J. Tabasco, the same Duroc sow used in the assembly of Sscrofa10.2. The PacBio reads were assembled using the Falcon assembly pipeline resulting in 3,206 contigs with an initial contig N50 of 14.5Mb. We used Sscrofa10.2 as a template to scaffold the PacBio contigs, under the assumption that its gross structure is correct, and used PBJelly to fill gaps. Additional gaps were filled using large, sequenced BACs from the original assembly. Following gap filling, the assembly has substantially improved contiguity and contains more sequence than the Sscrofa10.2 assembly. Arrow and Pilon were used to polish the assembly. The contig N50 is now 58.5Mb with 103 gaps remaining. By comparing regions of the two assemblies we show that regions with structural abnormalities we identified in Sscrofa10.2 are resolved in the new PacBio assembly.

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April 21, 2020  |  

Chlorella vulgaris genome assembly and annotation reveals the molecular basis for metabolic acclimation to high light conditions.

Chlorella vulgaris is a fast-growing fresh-water microalga cultivated at the industrial scale for applications ranging from food to biofuel production. To advance our understanding of its biology and to establish genetics tools for biotechnological manipulation, we sequenced the nuclear and organelle genomes of Chlorella vulgaris 211/11P by combining next generation sequencing and optical mapping of isolated DNA molecules. This hybrid approach allowed to assemble the nuclear genome in 14 pseudo-molecules with an N50 of 2.8 Mb and 98.9% of scaffolded genome. The integration of RNA-seq data obtained at two different irradiances of growth (high light-HL versus low light -LL) enabled to identify 10,724 nuclear genes, coding for 11,082 transcripts. Moreover 121 and 48 genes were respectively found in the chloroplast and mitochondrial genome. Functional annotation and expression analysis of nuclear, chloroplast and mitochondrial genome sequences revealed peculiar features of Chlorella vulgaris. Evidence of horizontal gene transfers from chloroplast to mitochondrial genome was observed. Furthermore, comparative transcriptomic analyses of LL vs HL provide insights into the molecular basis for metabolic rearrangement in HL vs. LL conditions leading to enhanced de novo fatty acid biosynthesis and triacylglycerol accumulation. The occurrence of a cytosolic fatty acid biosynthetic pathway can be predicted and its upregulation upon HL exposure is observed, consistent with increased lipid amount under HL. These data provide a rich genetic resource for future genome editing studies, and potential targets for biotechnological manipulation of Chlorella vulgaris or other microalgae species to improve biomass and lipid productivity.This article is protected by copyright. All rights reserved.

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April 21, 2020  |  

Chromosome-length haplotigs for yak and cattle from trio binning assembly of an F1 hybrid

Background Assemblies of diploid genomes are generally unphased, pseudo-haploid representations that do not correctly reconstruct the two parental haplotypes present in the individual sequenced. Instead, the assembly alternates between parental haplotypes and may contain duplications in regions where the parental haplotypes are sufficiently different. Trio binning is an approach to genome assembly that uses short reads from both parents to classify long reads from the offspring according to maternal or paternal haplotype origin, and is thus helped rather than impeded by heterozygosity. Using this approach, it is possible to derive two assemblies from an individual, accurately representing both parental contributions in their entirety with higher continuity and accuracy than is possible with other methods.Results We used trio binning to assemble reference genomes for two species from a single individual using an interspecies cross of yak (Bos grunniens) and cattle (Bos taurus). The high heterozygosity inherent to interspecies hybrids allowed us to confidently assign >99% of long reads from the F1 offspring to parental bins using unique k-mers from parental short reads. Both the maternal (yak) and paternal (cattle) assemblies contain over one third of the acrocentric chromosomes, including the two largest chromosomes, in single haplotigs.Conclusions These haplotigs are the first vertebrate chromosome arms to be assembled gap-free and fully phased, and the first time assemblies for two species have been created from a single individual. Both assemblies are the most continuous currently available for non-model vertebrates.MbmegabaseskbkilobasesMYAmillions of years agoMHCmajor histocompatibility complexSMRTsingle molecule real time

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April 21, 2020  |  

A robust benchmark for germline structural variant detection

New technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution, and comprehensiveness. Translating these methods to routine research and clinical practice requires robust benchmark sets. We developed the first benchmark set for identification of both false negative and false positive germline SVs, which complements recent efforts emphasizing increasingly comprehensive characterization of SVs. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle (GIAB) Consortium integrated 19 sequence-resolved variant calling methods, both alignment- and de novo assembly-based, from short-, linked-, and long-read sequencing, as well as optical and electronic mapping. The final benchmark set contains 12745 isolated, sequence-resolved insertion and deletion calls =50 base pairs (bp) discovered by at least 2 technologies or 5 callsets, genotyped as heterozygous or homozygous variants by long reads. The Tier 1 benchmark regions, for which any extra calls are putative false positives, cover 2.66 Gbp and 9641 SVs supported by at least one diploid assembly. Support for SVs was assessed using svviz with short-, linked-, and long-read sequence data. In general, there was strong support from multiple technologies for the benchmark SVs, with 90 % of the Tier 1 SVs having support in reads from more than one technology. The Mendelian genotype error rate was 0.3 %, and genotype concordance with manual curation was >98.7 %. We demonstrate the utility of the benchmark set by showing it reliably identifies both false negatives and false positives in high-quality SV callsets from short-, linked-, and long-read sequencing and optical mapping.

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April 21, 2020  |  

Large Fragment Deletions Induced by Cas9 Cleavage While Not in BEs System in Rabbit

CRISPR-Cas9 and BEs system are poised to become the gene editing tool of choice in clinical contexts, however large fragment deletion was found in Cas9-mediated mutation cells without animal level validation. By analyzing 16 gene-edited rabbit lines (including 112 rabbits) generated using SpCas9, BEs, xCas9 and xCas9-BEs with long-range PCR genotyping and long-read sequencing by PacBio platform, we show that extending thousands of bases fragment deletions in single-guide RNA/Cas9 and xCas9 system mutation rabbit, but few large deletions were found in BEs-induced mutation rabbits. We firstly validated that no large fragment deletion induced by BEs system at animal level, suggesting that BE systems can be beneficial tools for the further development of highly accurate and secure gene therapy for the clinical treatment of human genetic disorders

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April 21, 2020  |  

The Chinese chestnut genome: a reference for species restoration

Forest tree species are increasingly subject to severe mortalities from exotic pests, diseases, and invasive organisms, accelerated by climate change. Forest health issues are threatening multiple species and ecosystem sustainability globally. While sources of resistance may be available in related species, or among surviving trees, introgression of resistance genes into threatened tree species in reasonable time frames requires genome-wide breeding tools. Asian species of chestnut (Castanea spp.) are being employed as donors of disease resistance genes to restore native chestnut species in North America and Europe. To aid in the restoration of threatened chestnut species, we present the assembly of a reference genome with chromosome-scale sequences for Chinese chestnut (C. mollissima), the disease-resistance donor for American chestnut restoration. We also demonstrate the value of the genome as a platform for research and species restoration, including new insights into the evolution of blight resistance in Asian chestnut species, the locations in the genome of ecologically important signatures of selection differentiating American chestnut from Chinese chestnut, the identification of candidate genes for disease resistance, and preliminary comparisons of genome organization with related species.

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