June 1, 2021  |  

A novel analytical pipeline for de novo haplotype phasing and amplicon analysis using SMRT Sequencing technology.

While the identification of individual SNPs has been readily available for some time, the ability to accurately phase SNPs and structural variation across a haplotype has been a challenge. With individual reads of an average length of 9 kb (P5-C3), and individual reads beyond 30 kb in length, SMRT Sequencing technology allows the identification of mutation combinations such as microdeletions, insertions, and substitutions without any predetermined reference sequence. Long- amplicon analysis is a novel protocol that identifies and reports the abundance of differing clusters of sequencing reads within a single library. Graphs generated via hierarchical clustering of individual sequencing reads are used to generate Markov models representing the consensus sequence of individual clusters found to be significantly different. Long-amplicon analysis is capable of differentiating between underlying sequences that are 99.9% similar, which is suitable for haplotyping and differentiating pseudogenes from coding transcripts. This protocol allows for the identification of structural variation in the MUC5AC gene sequence, despite the presence of a gap in the current genome assembly, and can also be used for HLA haplotyping. Clustering can also been applied to identify full length transcripts for the purpose of estimating consensus sequences and enumerating isoform types. Long-amplicon analysis allows for the elucidation of complex regions otherwise missed by other sequencing technologies, which may contribute to the diagnosis and understanding of otherwise complex diseases.


June 1, 2021  |  

Resolving the ‘dark matter’ in genomes.

Second-generation sequencing has brought about tremendous insights into the genetic underpinnings of biology. However, there are many functionally important and medically relevant regions of genomes that are currently difficult or impossible to sequence, resulting in incomplete and fragmented views of genomes. Two main causes are (i) limitations to read DNA of extreme sequence content (GC-rich or AT-rich regions, low complexity sequence contexts) and (ii) insufficient read lengths which leave various forms of structural variation unresolved and result in mapping ambiguities.


June 1, 2021  |  

Full-length sequencing of HLA class I genes of more than 1000 samples provides deep insights into sequence variability

Aim: The vast majority of donor typing relies on sequencing exons 2 and 3 of HLA class I genes (HLA-A, -B, -C). With such an approach certain allele combinations do not result in the anticipated “high resolution” (G-code) typing, due to the lack of exon-phasing information. To resolve ambiguous typing results for a haplotype frequency project, we established a whole gene sequencing approach for HLA class I, facilitating also an estimation of the degree of sequence variability outside the commonly sequenced exons. Methods: Primers were developed flanking the UTR regions resulting in similar amplicon lengths of 4.2-4.4 kb. Using a 4-primer approach, secondary primers containing barcodes were combined with the gene specific primers to obtain barcoded full-gene amplicons in a single amplification step. Amplicons were pooled, purified, and ligated to SMRT bells (i.e. annealing points for sequencing primers) following standard protocols from Pacific Biosciences. Taking advantage of the SMRT chemistry, pools of 48-72 amplicons were sequenced full length and phased in single runs on a Pacific Biosciences RSII instrument. Demultiplexing was achieved using the SMRT portal. Sequence analysis was performed using NGSengine software (GenDx). Results: We successfully performed full-length gene sequencing of 1003 samples, harboring ambiguous typings of either HLA-A (n=46), HLA-B (n=304) or HLA-C (n=653). Despite the high per-read raw error rates typical for SMRT sequencing (~15%) the consensus sequence proved highly reliable. All consensus sequences for exons 2 and 3 were in full accordance with their MiSeq-derived sequences. Unambiguous allelic resolution was achieved for all samples. We observed novel intronic, exonic as well as UTR sequence variations for many of the alleles covered by our data set. This included sequences of 600 individuals with HLA-C*07:01/C*07:02 genotype revealing the extent of sequence variation outside the exons 2 and 3. Conclusion: Here we present a whole gene amplification and sequencing approach for HLA class I genes. The maturity of this approach was demonstrated by sequencing more than 1000 samples, achieving fully phased allelic sequences. Extensive sequencing of one common allele combination hints at the yet to discover diversity of the HLA system outside the commonly analyzed exons.


June 1, 2021  |  

Phased full-length SMRT Sequencing of HLA DPB1

Aim: In contrast to exon-based HLA-typing approaches, whole gene genotyping crucially depends on full-length sequences submitted to the IMGT/HLA Database. Currently, full-length sequences are provided for only 7 out of 520 HLA-DPB1 alleles. Therefore, we developed a fully phased whole-gene sequencing approach for DPB1, to facilitate further exploration of the allelic structure at this locus. Methods: Primers were developed flanking the UTR-regions of DPB1 resulting in a 12 kb amplicon. Using a 4-primer approach, secondary primers containing barcodes were combined with the gene-specific primers to obtain barcoded full-gene amplicons in a single amplification step. Amplicons were pooled, purified, and ligated to SMRT bells (i.e. annealing points for sequencing primers) following standard protocols from Pacific Biosciences. Taking advantage of the SMRT chemistry, pools of 48 amplicons were sequenced full length in single runs on a Pacific Biosciences RSII instrument. Demultiplexing was performed using the SMRT portal. Sequence analysis was performed using the NGSengine software (GenDx). Results: We analyzed a set of 48 randomly picked samples. With 3 exceptions due to PCR failure, all genotype assignments conformed to standard genotyping results based on exons 2 and 3. Allelic proportions for heterozygous positions were evenly distributed (range 0.4 – 0.6) for all samples, suggesting unbiased amplifications. Despite the high per-read raw error rates typical for SMRT sequencing (~15%) the consensus sequence proved highly reliable. All consensus sequences for exons 2 and 3 were in full accordance with their MiSeq-derived sequences. We describe novel intronic sequence variation of the 7 so far genomically defined alleles, as well as 7 whole-length DPB1 alleles with hitherto unknown intronic regions. One of these alleles (HLA-DPB1*131:01) is classified as rare. Conclusion: Here we present a whole gene amplification and sequencing workflow for DPB1 alleles utilizing single molecule real-time (SMRT) sequencing from Pacific Biosciences. Validation of consensus sequences against known exonic sequences highlights the reliability of this technology. This workflow will facilitate amending the IMGT/HLA Database for DPB1.


June 1, 2021  |  

Characterizing haplotype diversity at the immunoglobulin heavy chain locus across human populations using novel long-read sequencing and assembly approaches

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 antibody repertoire variability and disease. To remedy this, we are taking a multi-faceted approach to improving existing genomic resources in the human IGH region. First, from whole-genome and fosmid-based datasets, we are building the largest and most ethnically diverse set of IGH reference assemblies to date, by employing PacBio long-read sequencing combined with novel algorithms for phased haplotype assembly. In total, our effort will result in the characterization of >15 phased haplotypes from individuals of Asian, African, and European descent, to be used as a representative reference set by the genomics and immunogenetics community. Second, we are utilizing this more comprehensive sequence catalogue to inform the design and analysis of novel targeted IGH genotyping assays. Standard targeted DNA enrichment methods (e.g., exome capture) are currently optimized for the capture of only very short (100’s of bp) DNA segments. Our platform uses a modified bench protocol to pair existing capture-array technologies with the enrichment of longer fragments of DNA, enabling the use of PacBio sequencing of DNA segments up to 7 Kb. This substantial increase in contiguity disambiguates many of the complex repeated structures inherent to the locus, while yielding the base pair fidelity required to call SNVs. Together these resources will establish a stronger framework for further characterizing IGH genetic diversity and facilitate IGH genomic profiling in the clinical and research settings, which will be key to fully understanding the role of IGH germline variation in antibody repertoire development and disease.


June 1, 2021  |  

No-amp targeted SMRT sequencing using a CRISPR-Cas9 enrichment method

Targeted sequencing of genomic DNA requires an enrichment method to generate detectable amounts of sequencing products. Genomic regions with extreme composition bias and repetitive sequences can pose a significant enrichment challenge. Many genetic diseases caused by repeat element expansions are representative of these challenging enrichment targets. PCR amplification, used either alone or in combination with a hybridization capture method, is a common approach for target enrichment. While PCR amplification can be used successfully with genomic regions of moderate to high complexity, it is the low-complexity regions and regions containing repetitive elements sometimes of indeterminate lengths due to repeat expansions that can lead to poor or failed PCR enrichment. We have developed an enrichment method for targeted SMRT Sequencing on the PacBio Sequel System using the CRISPR-Cas9 system that requires no PCR amplification. Briefly, a preformed SMRTbell library containing the target region of interest is cleaved with Cas9 through direct interaction with a sequence-specific guide RNA. After ligation with new poly(A) hairpin adapters, the asymmetric SMRTbell templates are enriched by magnetic bead separation. This method, paired with SMRT Sequencing’s long reads, high consensus accuracy, and uniform coverage, allows sequencing of genomic regions regardless of challenging sequence context that cannot be investigated with other technologies. The method is amenable to analyzing multiple samples and/or targets in a single reaction. In addition, this method also preserves epigenetic modifications allowing for the detection and characterization of DNA methylation which has been shown to be a key factor in the disease mechanism for some repeat expansion diseases. Here we present results of our latest No-Amp Targeted Sequencing procedure applied to the characterization of CAG triplet repeat expansions in the HTT gene responsible for Huntington’s Disease.


April 21, 2020  |  

Acquired N-Linked Glycosylation Motifs in B-Cell Receptors of Primary Cutaneous B-Cell Lymphoma and the Normal B-Cell Repertoire.

Primary cutaneous follicle center lymphoma (PCFCL) is a rare mature B-cell lymphoma with an unknown etiology. PCFCL resembles follicular lymphoma (FL) by cytomorphologic and microarchitectural criteria. FL B cells are selected for N-linked glycosylation motifs in their B-cell receptors (BCRs) that are acquired during continuous somatic hypermutation. The stimulation of mannosylated BCR by lectins on the tumor microenvironment is therefore a candidate driver in FL pathogenesis. We investigated whether the same mechanism could play a role in PCFCL pathogenesis. Full-length functional variable, diversity, and joining gene sequences of 18 PCFCL and 8 primary cutaneous diffuse large B-cell lymphoma, leg-type were identified by unbiased Anchoring Reverse Transcription of Immunoglobulin Sequences and Amplification by Nested PCR and BCR reconstruction from RNA sequencing data. Low BCR variation demonstrated negligible ongoing somatic hypermutation in PCFCL and primary cutaneous diffuse large B-cell lymphoma, leg-type, and indicated that the PCFCL microarchitecture does not act as a functional germinal center. Similar to FL but in contrast to primary cutaneous diffuse large B-cell lymphoma, leg-type, BCR genes of 15 PCFCLs (83%) had acquired N-linked glycosylation motifs. These motifs were located at the BCR positions converted to N-linked glycosylation motifs in normal B-cell repertoires with low prevalence but mostly at different positions than those found in FL. The cutaneous localization of PCFCL might suggest a role for lectins from commensal skin bacteria in PCFCL lymphomagenesis.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.


April 21, 2020  |  

Benchmarking Transposable Element Annotation Methods for Creation of a Streamlined, Comprehensive Pipeline

Sequencing technology and assembly algorithms have matured to the point that high-quality de novo assembly is possible for large, repetitive genomes. Current assemblies traverse transposable elements (TEs) and allow for annotation of TEs. There are numerous methods for each class of elements with unknown relative performance metrics. We benchmarked existing programs based on a curated library of rice TEs. Using the most robust programs, we created a comprehensive pipeline called Extensive de-novo TE Annotator (EDTA) that produces a condensed TE library for annotations of structurally intact and fragmented elements. EDTA is open-source and freely available: https://github.com/oushujun/EDTA.List of abbreviationsTETransposable ElementsLTRLong Terminal RepeatLINELong Interspersed Nuclear ElementSINEShort Interspersed Nuclear ElementMITEMiniature Inverted Transposable ElementTIRTerminal Inverted RepeatTSDTarget Site DuplicationTPTrue PositivesFPFalse PositivesTNTrue NegativeFNFalse NegativesGRFGeneric Repeat FinderEDTAExtensive de-novo TE Annotator


April 21, 2020  |  

Genome-wide selection footprints and deleterious variations in young Asian allotetraploid rapeseed.

Brassica napus (AACC, 2n = 38) is an important oilseed crop grown worldwide. However, little is known about the population evolution of this species, the genomic difference between its major genetic groups, such as European and Asian rapeseed, and the impacts of historical large-scale introgression events on this young tetraploid. In this study, we reported the de novo assembly of the genome sequences of an Asian rapeseed (B. napus), Ningyou 7, and its four progenitors and compared these genomes with other available genomic data from diverse European and Asian cultivars. Our results showed that Asian rapeseed originally derived from European rapeseed but subsequently significantly diverged, with rapid genome differentiation after hybridization and intensive local selective breeding. The first historical introgression of B. rapa dramatically broadened the allelic pool but decreased the deleterious variations of Asian rapeseed. The second historical introgression of the double-low traits of European rapeseed (canola) has reshaped Asian rapeseed into two groups (double-low and double-high), accompanied by an increase in genetic load in the double-low group. This study demonstrates distinctive genomic footprints and deleterious SNP (single nucleotide polymorphism) variants for local adaptation by recent intra- and interspecies introgression events and provides novel insights for understanding the rapid genome evolution of a young allopolyploid crop. © 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.


April 21, 2020  |  

Early Sex-chromosome Evolution in the Diploid Dioecious Plant Mercurialis annua.

Suppressed recombination allows divergence between homologous sex chromosomes and the functionality of their genes. Here, we reveal patterns of the earliest stages of sex-chromosome evolution in the diploid dioecious herb Mercurialis annua on the basis of cytological analysis, de novo genome assembly and annotation, genetic mapping, exome resequencing of natural populations, and transcriptome analysis. The genome assembly contained 34,105 expressed genes, of which 10,076 were assigned to linkage groups. Genetic mapping and exome resequencing of individuals across the species range both identified the largest linkage group, LG1, as the sex chromosome. Although the sex chromosomes of M. annua are karyotypically homomorphic, we estimate that about a third of the Y chromosome has ceased recombining, containing 568 transcripts and spanning 22.3 cM in the corresponding female map. Nevertheless, we found limited evidence for Y-chromosome degeneration in terms of gene loss and pseudogenization, and most X- and Y-linked genes appear to have diverged in the period subsequent to speciation between M. annua and its sister species M. huetii which shares the same sex-determining region. Taken together, our results suggest that the M. annua Y chromosome has at least two evolutionary strata: a small old stratum shared with M. huetii, and a more recent larger stratum that is probably unique to M. annua and that stopped recombining about one million years ago. Patterns of gene expression within the non-recombining region are consistent with the idea that sexually antagonistic selection may have played a role in favoring suppressed recombination.Copyright © 2019, Genetics.


April 21, 2020  |  

A Highly Unusual V1 Region of Env in an Elite Controller of HIV Infection.

HIV elite controllers represent a remarkable minority of patients who maintain normal CD4+ T-cell counts and low or undetectable viral loads for decades in the absence of antiretroviral therapy. To examine the possible contribution of virus attenuation to elite control, we obtained a primary HIV-1 isolate from an elite controller who had been infected for 19?years, the last 10 of which were in the absence of antiretroviral therapy. Full-length sequencing of this isolate revealed a highly unusual V1 domain in Envelope (Env). The V1 domain in this HIV-1 strain was 49 amino acids, placing it in the top 1% of lengths among the 6,112 Env sequences in the Los Alamos National Laboratory online database. Furthermore, it included two additional N-glycosylation sites and a pair of cysteines suggestive of an extra disulfide loop. Virus with this Env retained good infectivity and replicative capacity; however, analysis of recombinant viruses suggested that other sequences in Env were adapted to accommodate the unusual V1 domain. While the long V1 domain did not confer resistance to neutralization by monoclonal antibodies of the V1/V2-glycan-dependent class, it did confer resistance to neutralization by monoclonal antibodies of the V3-glycan-dependent class. Our findings support results in the literature that suggest a role for long V1 regions in shielding HIV-1 from recognition by V3-directed broadly neutralizing antibodies. In the case of the elite controller described here, it seems likely that selective pressures from the humoral immune system were responsible for driving the highly unusual polymorphisms present in this HIV-1 Envelope.IMPORTANCE Elite controllers have long provided an avenue for researchers to reveal mechanisms underlying control of HIV-1. While the role of host genetic factors in facilitating elite control is well known, the possibility of infection by attenuated strains of HIV-1 has been much less studied. Here we describe an unusual viral feature found in an elite controller of HIV-1 infection and demonstrate its role in conferring escape from monoclonal antibodies of the V3-glycan class. Our results suggest that extreme variation may be needed by HIV-1 to escape neutralization by some antibody specificities. Copyright © 2019 Silver et al.


April 21, 2020  |  

Klebsiella quasipneumoniae Provides a Window into Carbapenemase Gene Transfer, Plasmid Rearrangements, and Patient Interactions with the Hospital Environment.

Several emerging pathogens have arisen as a result of selection pressures exerted by modern health care. Klebsiella quasipneumoniae was recently defined as a new species, yet its prevalence, niche, and propensity to acquire antimicrobial resistance genes are not fully described. We have been tracking inter- and intraspecies transmission of the Klebsiella pneumoniae carbapenemase (KPC) gene, blaKPC, between bacteria isolated from a single institution. We applied a combination of Illumina and PacBio whole-genome sequencing to identify and compare K. quasipneumoniae from patients and the hospital environment over 10- and 5-year periods, respectively. There were 32 blaKPC-positive K. quasipneumoniae isolates, all of which were identified as K. pneumoniae in the clinical microbiology laboratory, from 8 patients and 11 sink drains, with evidence for seven separate blaKPC plasmid acquisitions. Analysis of a single subclade of K. quasipneumoniae subsp. quasipneumoniae (n?=?23 isolates) from three patients and six rooms demonstrated seeding of a sink by a patient, subsequent persistence of the strain in the hospital environment, and then possible transmission to another patient. Longitudinal analysis of this strain demonstrated the acquisition of two unique blaKPC plasmids and then subsequent within-strain genetic rearrangement through transposition and homologous recombination. Our analysis highlights the apparent molecular propensity of K. quasipneumoniae to persist in the environment as well as acquire carbapenemase plasmids from other species and enabled an assessment of the genetic rearrangements which may facilitate horizontal transmission of carbapenemases. Copyright © 2019 Mathers et al.


April 21, 2020  |  

Centromeric Satellite DNAs: Hidden Sequence Variation in the Human Population.

The central goal of medical genomics is to understand the inherited basis of sequence variation that underlies human physiology, evolution, and disease. Functional association studies currently ignore millions of bases that span each centromeric region and acrocentric short arm. These regions are enriched in long arrays of tandem repeats, or satellite DNAs, that are known to vary extensively in copy number and repeat structure in the human population. Satellite sequence variation in the human genome is often so large that it is detected cytogenetically, yet due to the lack of a reference assembly and informatics tools to measure this variability, contemporary high-resolution disease association studies are unable to detect causal variants in these regions. Nevertheless, recently uncovered associations between satellite DNA variation and human disease support that these regions present a substantial and biologically important fraction of human sequence variation. Therefore, there is a pressing and unmet need to detect and incorporate this uncharacterized sequence variation into broad studies of human evolution and medical genomics. Here I discuss the current knowledge of satellite DNA variation in the human genome, focusing on centromeric satellites and their potential implications for disease.


April 21, 2020  |  

Chromosome-scale assemblies reveal the structural evolution of African cichlid genomes.

African cichlid fishes are well known for their rapid radiations and are a model system for studying evolutionary processes. Here we compare multiple, high-quality, chromosome-scale genome assemblies to elucidate the genetic mechanisms underlying cichlid diversification and study how genome structure evolves in rapidly radiating lineages.We re-anchored our recent assembly of the Nile tilapia (Oreochromis niloticus) genome using a new high-density genetic map. We also developed a new de novo genome assembly of the Lake Malawi cichlid, Metriaclima zebra, using high-coverage Pacific Biosciences sequencing, and anchored contigs to linkage groups (LGs) using 4 different genetic maps. These new anchored assemblies allow the first chromosome-scale comparisons of African cichlid genomes. Large intra-chromosomal structural differences (~2-28 megabase pairs) among species are common, while inter-chromosomal differences are rare (<10 megabase pairs total). Placement of the centromeres within the chromosome-scale assemblies identifies large structural differences that explain many of the karyotype differences among species. Structural differences are also associated with unique patterns of recombination on sex chromosomes. Structural differences on LG9, LG11, and LG20 are associated with reduced recombination, indicative of inversions between the rock- and sand-dwelling clades of Lake Malawi cichlids. M. zebra has a larger number of recent transposable element insertions compared with O. niloticus, suggesting that several transposable element families have a higher rate of insertion in the haplochromine cichlid lineage.This study identifies novel structural variation among East African cichlid genomes and provides a new set of genomic resources to support research on the mechanisms driving cichlid adaptation and speciation. © The Author(s) 2019. Published by Oxford University Press.


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