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…
Purpose: Clinical laboratories, research laboratories and technology developers all need DNA samples with reliably known genotypes in order to help validate and improve their methods. The Genome in a Bottle Consortium (genomeinabottle.org) has been developing Reference Materials with high-accuracy whole genome sequences to support these efforts.Methodology: Our pilot reference material is based on Coriell sample NA12878 and was released in May 2015 as NIST RM 8398 (tinyurl.com/giabpilot). To minimize bias and improve accuracy, 11 whole-genome and 3 exome data sets produced using 5 different technologies were integrated using a systematic arbitration method [1]. The Genome in a Bottle Analysis Group…
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),…
The complex immune regions of the genome, including MHC and KIR, contain large copy number variants (CNVs), a high density of genes, hyper-polymorphic gene alleles, and conserved extended haplotypes (CEH) with enormous linkage disequilibrium (LDs). This level of complexity and inherent biases of short-read sequencing make it challenging for extracting immune region haplotype information from reference-reliant, shotgun sequencing and GWAS methods. As NGS based genome and exome sequencing and SNP arrays have become a routine for population studies, numerous efforts are being made for developing software to extract and or impute the immune gene information from these datasets. Despite these…
The killer immunoglobulin-like receptors (KIR) genes belong to the immunoglobulin superfamily and are widely studied due to the critical role they play in coordinating the innate immune response to infection and disease. Highly accurate, contiguous, long reads, like those generated by SMRT Sequencing, when combined with target-enrichment protocols, provide a straightforward strategy for generating complete de novo assembled KIR haplotypes. We have explored two different methods to capture the KIR region; one applying the use of fosmid clones and one using Nimblegen capture.
The increased sequencing throughput creates a need for multiplexing for several applications. We are here detailing different barcoding strategies for microbial sequencing, targeted sequencing, Iso-Seq full-length isoform sequencing, and Roche NimbleGen’s target enrichment method.
The long reads, random error, and unbiased sampling of SMRT Sequencing enables high quality, de novo assembly of the human genome. PacBio long reads are capable of resolving genomic variations at all size scales, including SNPs, insertions, deletions, inversions, translocations, and repeat expansions, all of which are both important in understanding the genetic basis for human disease, and difficult to access via other technologies. In demonstration of this, we report a new high-quality, diploid-aware de novo assembly of Craig Venter’s well-studied genome.
Genes associated with several neurological disorders have been shown to be highly polymorphic. Targeted sequencing of these genes using NGS technologies is a powerful way to increase the cost-effectiveness of variant discovery and detection. However, for a comprehensive view of these target genes, it is necessary to have complete and uniform coverage across regions of interest. Unfortunately, short-read sequencing technologies are not ideal for these types of studies as they are prone to mis-mapping and often fail to span repetitive regions. Targeted sequencing with PacBio long reads provides the unique advantage of single-molecule observations of complex genomic regions. PacBio long…
Alzheimer’s disease (AD) is a devastating neurodegenerative disease that is genetically complex. Although great progress has been made in identifying fully penetrant mutations in genes such as APP, PSEN1 and PSEN2 that cause early-onset AD, these still represent a very small percentage of AD cases. Large-scale, genome-wide association studies (GWAS) have identified at least 20 additional genetic risk loci for the more common form of late-onset AD. However, the identified SNPs are typically not the actual risk variants, but are in linkage disequilibrium with the presumed causative variant (Van Cauwenberghe C, et al., The genetic landscape of Alzheimer disease: clinical…
In recent years, human genomic research has focused on comparing short-read data sets to a single human reference genome. However, it is becoming increasingly clear that significant structural variations present in individual human genomes are missed or ignored by this approach. Additionally, remapping short-read data limits the phasing of variation among individual chromosomes. This reduces the newly sequenced genome to a table of single nucleotide polymorphisms (SNPs) with little to no information as to the co-linearity (phasing) of these variants, resulting in a “mosaic” reference representing neither of the parental chromosomes. The variation between the homologous chromosomes is lost in…
Nucleotide repeat expansions are a major cause of neurological and neuromuscular disease in humans, however, the nature of these genomic regions makes characterizing them extremely challenging. Accurate DNA sequencing of repeat expansions using short-read sequencing technologies is difficult, as short-read technologies often cannot read through regions of low sequence complexity. Additionally, these short reads do not span the entire region of interest and therefore sequence assembly is required. Lastly, most target enrichment methods are reliant upon amplification which adds the additional caveat of PCR bias. We have developed a novel, amplification-free enrichment technique that employs the CRISPR/Cas9 system for specific…
The human immunoglobulin heavy chain locus (IGH) remains among the most understudied regions of the human genome. Recent efforts have shown that haplotype diversity within IGH is elevated and exhibits population specific patterns; for example, our re-sequencing of the locus from only a single chromosome uncovered >100 Kb of novel sequence, including descriptions of six novel alleles, and four previously unmapped genes. Historically, this complex locus architecture has hindered the characterization of IGH germline single nucleotide, copy number, and structural variants (SNVs; CNVs; SVs), and as a result, there remains little known about the role of IGH polymorphisms in inter-individual…
Structural variant calling combining Illumina and low-coverage Pacbio Detection of large genomic variation (structural variants) has proven challenging using short-read methods. Long-read approaches which can span these large events have promise to dramatically expand the ability to accurately call structural variants. Although sequencing with Pacific Biosciences (Pacbio) long-read technology has become increasingly high throughput, generating high coverage with the technology can still be limiting and investigators often would like to know what pacbio coverages are adequate to call structural variants. Here, we present a method to identify a substantially higher fraction of structural variants in the human genome using low-coverage…
Over 40% of males and ~16% of female carriers of a FMR1 premutation allele (55-200 CGG repeats) are at risk for developing Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), an adult onset neurodegenerative disorder while, about 20% of female carriers will develop Fragile X-associated Primary Ovarian Insufficiency (FXPOI), in addition to a number of adult-onset clinical problems (FMR1 associated disorders). Marked elevation in FMR1 mRNA levels have been observed with premutation alleles and the resulting RNA toxicity is believed to be the leading molecular mechanism proposed for these disorders. The FMR1 gene, as many housekeeping genes, undergoes alternative splicing. Using long-read isoform…