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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.

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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.

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June 1, 2021  |  

Access full spectrum of polymorphisms in HLA class I & II genes, without imputation for disease association and evolutionary research.

MHC class I and II genes are critically monitored by high-resolution sequencing for organ transplant decisions due to their role in GVHD. Their direct or linkage-based causal association, have increased their prominence as targets for drug sensitivity, autoimmune, cancer and infectious disease research. Monitoring HLA genes can however be tricky due to their highly polymorphic nature. Allele-level resolution is thus strongly preferred. However, most studies were historically focused on peptide binding domains of the HLA genes, due to technological challenges. As a result knowledge about the functional role of polymorphisms outside of exons 2 and 3 of HLA genes was rather limited. There are also relatively few full-length gene references currently available in the IMGT HLA database. This made it difficult to quickly adopt high-throughput reference-reliant methods for allele-level HLA sequencing. Increasing awareness regarding role of regulatory region polymorphisms of HLA genes in disease association1, nonetheless have brought about a revolution in full-length HLA gene sequencing. Researchers are now exploring ways to obtain complete information for HLA genes and integrate it with the current HLA database so it can be interpreted used by clinical researchers. We have explored advantages of SMRT Sequencing to obtain fully phased, allele-specific sequences of HLA class I and II genes for 96 samples using completely De novo consensus generation approach for imputation-free 4-field typing. With long read lengths (average >10 kb) and consensus accuracy exceeding 99.999% (Q50), a comprehensive snapshot of variants in exons, introns and UTRs could be obtained for spectrum of polymorphisms in phase across SNP-poor regions. Such information can provide invaluable insights in future causality association and population diversity research.

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June 1, 2021  |  

Full-length cDNA sequencing of alternatively spliced isoforms provides insight into human cancer

The majority of human genes are alternatively spliced, making it possible for most genes to generate multiple proteins. The process of alternative splicing is highly regulated in a developmental-stage and tissue-specific manner. Perturbations in the regulation of these events can lead to disease in humans (1). Alternative splicing has been shown to play a role in human cancer, muscular dystrophy, Alzheimer’s, and many other diseases. Understanding these diseases requires knowing the full complement of mRNA isoforms. Microarrays and high-throughput cDNA sequencing have become highly successful tools for studying transcriptomes, however these technologies only provide small fragments of transcripts and building complete transcript isoforms has been very challenging (2). We have developed a technique, called Iso-Seq sequencing, that is capable of sequencing full-length, single-molecule cDNA sequences. The method employs SMRT Sequencing from PacBio, which can sequence individual molecules with read lengths that average more than 10 kb and can reach as long as 40 kb. As most transcripts are from 1 – 10 kb, we can sequence through entire RNA molecules, requiring no fragmentation or post-sequencing assembly. Jointly with the sequencing method, we developed a computational pipeline that polishes these full-length transcript sequences into high-quality, non-redundant transcript consensus sequences. Iso-Seq sequencing enables unambiguous identification of alternative splicing events, alternative transcriptional start and polyA sites, and transcripts from gene fusion events. Knowledge of the complete set of isoforms from a sample of interest is key for accurate quantification of isoform abundance when using any technology for transcriptome studies (3). Here we characterize the full-length transcriptome of paired tumor/normal samples from breast cancer using deep Iso-Seq sequencing. We highlight numerous discoveries of novel alternatively spliced isoforms, gene-fusion events, and previously unannotated genes that will improve our understanding of human cancer. (1) Faustino NA and Cooper TA. Genes and Development. 2003. 17: 419-437(2) Steijger T, et al. Nat Methods. 2013 Dec;10(12):1177-84.(3) Au KF, et al. Proc Natl Acad Sci U S A. 2013 Dec 10;110(50):E4821-30.

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June 1, 2021  |  

Epigenome characterization of human genomes using the PacBio platform

In addition to the genome and transcriptome, epigenetic information is essential to understand biological processes and their regulation, and their misregulation underlying disease. Traditionally, epigenetic DNA modifications are detected using upfront sample preparation steps such as bisulfite conversion, followed by sequencing. Bisulfite sequencing has provided a wealth of knowledge about human epigenetics, however it does not access the entire genome due to limitations in read length and GC- bias of the sequencing technologies used. In contrast, Single Molecule, Real-Time (SMRT) DNA Sequencing is unique in that it can detect DNA base modifications as part of the sequencing process. It can thereby leverage the long read lengths and lack of GC bias for more comprehensive views of the human epigenome. I will highlight several examples of this capability towards the generation of new biological insights, including the resolution of methylation states in repetitive and GC-rich regions of the genome, and large-scale changes in the methylation status across a cancer genome as a function of drug sensitivity.

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June 1, 2021  |  

Highly sensitive and cost-effective detection of BRCA1 and BRCA2 cancer variants in FFPE samples using Multiplicom’s MASTR technology & Single Molecule, Real-Time (SMRT) Sequencing

Specific mutations in BRCA1 and BRCA2 have been shown to be associated with several types of cancers. Molecular profiling of cancer samples requires assays capable of accurately detecting the entire spectrum of variants, including those at relatively low frequency. Next-Generation Sequencing (NGS) has been a powerful tool for researchers to better understand cancer genetics. Here we describe a targeted re-sequencing workflow that combines barcoded amplification of BRCA1 and BRCA2 exons from 12 FFPE tumor samples using Multiplicom’s MASTR technology with PacBio SMRT Sequencing. This combination allows for the accurate detection of variants in a cost-effective and timely manner.

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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  |  

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  |  

Genome and transcriptome of the refeneration-competent flatworm, Macrostomum lignano

The free-living flatworm, Macrostomum lignano, much like its better known planarian relative, Schmidtea mediterranea, has an impressive regenerative capacity. Following injury, this species has the ability to regenerate almost an entirely new organism. This is attributable to the presence of an abundant somatic stem cell population, the neoblasts. These cells are also essential for the ongoing maintenance of most tissues, as their loss leads to irreversible degeneration of the animal. This set of unique properties makes a subset of flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cell fate specification, and regeneration. The use of these organisms as models, however, is hampered by the lack of a well-assembled and annotated genome sequences, fundamental to modern genetic and molecular studies. Here we report the genomic sequence of Macrostomum lignano and an accompanying characterization of its transcriptome. The genome structure of M. lignano is remarkably complex, with ~75% of its sequence being comprised of simple repeats and transposon sequences. This has made high quality assembly from Illumina reads alone impossible (N50=222 bp). We therefore generated 130X coverage by long sequencing reads from the PacBio platform to create a substantially improved assembly with an N50 of 64 Kbp. We complemented the reference genome with an assembled and annotated transcriptome, and used both of these datasets in combination to probe gene expression patterns during regeneration, examining pathways important to stem cell function. As a whole, our data will provide a crucial resource for the community for the study not only of invertebrate evolution and phylogeny but also of regeneration and somatic pluripotency.

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June 1, 2021  |  

The resurgence of reference quality genome

Several new 3rd generation long-range DNA sequencing and mapping technologies have recently become available that are starting to create a resurgence in genome sequence quality. Unlike their 2nd generation, shortread counterparts that can resolve a few hundred or a few thousand basepairs, the new technologies can routinely sequence 10,000 bp reads or map across 100,000 bp molecules. The substantially greater lengths are being used to enhance a number of important problems in genomics and medicine, including de novo genome assembly, structural variation detection, and haplotype phasing. Here we discuss the capabilities of the latest technologies, and show how they will improve the “3Cs of Genome Assembly”: the contiguity, completeness, and correctness. We derive this analysis from (1) a metaanalysis of the currently available 3rd generation genome assemblies, (2) a retrospective analysis of the evolution of the reference human genome, and (3) extensive simulations with dozens of species across the tree of life. We also propose a model using support vector regression (SVR) that predicts genome assembly performance using four features: read lengths(L) and coverage values(C) that can be used for evaluating potential technologies along with genome size(G) and repeats(R) that present species specific characteristics. The proposed model significantly improves genome assembly performance prediction by adopting data-driven approach and addressing limitations of the previous hypothesis-driven methodology. Overall, we anticipate these technologies unlock the genomic “dark matter”, and provide many new insights into evolution, agriculture, and human diseases.

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June 1, 2021  |  

Profiling metagenomic communities using circular consensus and Single Molecule, Real-Time Sequencing

There are many sequencing-based approaches to understanding complex metagenomic communities, spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR amplification. Whole-sample shotgun experiments require a high depth of coverage. As such, rare community members may not be represented in the resulting assembly. Circular-consensus, Single Molecule, Real-Time (SMRT) Sequencing reads in the 1-2 kb range, with >99% consensus accuracy, can be efficiently generated for low amounts of input DNA, e.g. as little as 10 ng of input DNA sequenced in 4 SMRT Cells can generate >100,000 such reads. While throughput is low compared to second-generation sequencing, the reads are a true random sampling of the underlying community. Long read lengths translate to a high number of the reads harboring full genes or even full operons for downstream analysis. Here we present the results of circular-consensus sequencing on a mock metagenomic community with an abundance range of multiple orders of magnitude, and compare the results with both 16S and shotgun assembly methods. We show that even with relatively low sequencing depth, the long-read, assembly-free, random sampling allows to elucidate meaningful information from the very low-abundance community members. For example, given the above low-input sequencing approach, a community member at 1/1,000 relative abundance would generate 100 1-2 kb sequence fragments having 99% consensus accuracy, with a high probability of containing a gene fragment useful for taxonomic classification or functional insight.

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June 1, 2021  |  

SMRT Sequencing of the alala genome

Single Molecule Real-Time (SMRT) Sequencing was used to generate long reads for whole genome shotgun sequencing of the genome of the`alala (Hawaiian crow). The ‘alala is endemic to Hawaii, and the only surviving lineage of the crow family, Corvidae, in the Hawaiian Islands. The population declined to less than 20 individuals in the 1990s, and today this charismatic species is extinct in the wild. Currently existing in only two captive breeding facilities, reintroduction of the ‘alala is scheduled to begin in the Fall of 2016. Reintroduction efforts will be assisted by information from the ‘alala genome generated and assembled by SMRT Technology, which will allow detailed analysis of genes associated with immunity, behavior, and learning. Using SMRT Sequencing, we present here best practices for achieving long reads for whole genome shotgun sequencing for complex plant and animal genomes such as the ‘alala genome. With recent advances in SMRTbell library preparation, P6-C4 chemistry and 6-hour movies, the number of useable bases now exceeds 1 Gb per SMRT Cell. Read lengths averaging 10 – 15 kb can be routinely achieved, with the longest reads approaching 70 kb. Furthermore, > 25% of useable bases are in reads greater than 30 kb, advantageous for generating contiguous draft assemblies of contig N50 up to 5 Mb. De novo assemblies of large genomes are now more tractable using SMRT Sequencing as the standalone technology. We also present guidelines for planning out projects for the de novo assembly of large genomes.

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June 1, 2021  |  

Cogent: Reconstructing the coding genome from full-length transcriptome sequences

For highly complex and large genomes, a well-annotated genome may be computationally challenging and costly, yet the study of alternative splicing events and gene annotations usually rely on the existence of a genome. Long-read sequencing technology provides new opportunities to sequence full-length cDNAs, avoiding computational challenges that short read transcript assembly brings. The use of single molecule, real-time sequencing from Pacific Biosciences to sequence transcriptomes (the Iso-SeqTM method), which produces de novo, high-quality, full-length transcripts, has revealed an astonishing amount of alternative splicing in eukaryotic species. With the Iso-Seq method, it is now possible to reconstruct the transcribed regions of the genome using just the transcripts themselves. We present Cogent, a tool for finding gene families and reconstructing the coding genome in the absence of a reference genome. Cogent uses k-mer similarities to first partition the transcripts into different gene families. Then, for each gene family, the transcripts are used to build a splice graph. Cogent identifies bubbles resulting from sequencing errors, minor variants, and exon skipping events, and attempts to resolve each splice graph down to the minimal set of reconstructed contigs. We apply Cogent to a Cuttlefish Iso-Seq dataset, for which there is a highly fragmented, Illumina-based draft genome assembly and little annotation. We show that Cogent successfully discovers gene families and can reconstruct the coding region of gene loci. The reconstructed contigs can then be used to visualize alternative splicing events, identify minor variants, and even be used to improve genome assemblies.

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June 1, 2021  |  

Full-length cDNA sequencing for genome annotation and analysis of alternative splicing

In higher eukaryotic organisms, the majority of multi-exon genes are alternatively spliced. Different mRNA isoforms from the same gene can produce proteins that have distinct properties and functions. Thus, the importance of understanding the full complement of transcript isoforms with potential phenotypic impact cannot be understated. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. The Iso-Seq protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences to survey transcriptome isoform diversity useful for gene discovery and annotation. Knowledge of the complete isoform repertoire is also key for accurate quantification of isoform abundance. As most transcripts range from 1 – 10 kb, fully intact RNA molecules can be sequenced using SMRT Sequencing without requiring fragmentation or post-sequencing assembly. Our open-source computational pipeline delivers high-quality, non-redundant sequences for unambiguous identification of alternative splicing events, alternative transcriptional start sites, polyA tail, and gene fusion events. We applied the Iso-Seq method to the maize (Zea mays) inbred line B73. Full-length cDNAs from six diverse tissues were barcoded and sequenced across multiple size-fractionated SMRTbell libraries. A total of 111,151 unique transcripts were identified. More than half of these transcripts (57%) represented novel, sometimes tissue-specific, isoforms of known genes. In addition to the 2250 novel coding genes and 860 lncRNAs discovered, the Iso-Seq dataset corrected errors in existing gene models, highlighting the value of full-length transcripts for whole gene annotations.

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