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June 1, 2021

Structural variant combining Illumina and low-coverage PacBio

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 pacbio data by multiple strategies for ensembling data types and algorithms. Algorithmically, we combine three structural variant callers: PBHoney by Adam English, Sniffles by Fritz Sedlazeck, and Parliament by Adam English (which we have modified to improve for speed). Parliament itself uses a combination of Pacbio and Illumina data with a number of short-read callers (Breakdancer, Pindel, Crest, CNVnator, Delly, and Lumpy). We show that the outputs of these three programs are largely complementary to each other, with each able to uniquely access different sets of structural variants at different coverages. Combining them together can more than double the recall of true structural variants from a truth set relative to sequencing with Illumina alone, with substantial improvements even at low pacbio coverages (3x – 7x). This allows us to present for the first time cost-benefit tradeoffs to investigators about how much pacbio sequencing will yield what improvements in SV-calling. This work also builds upon the foundational work of Genome in a Bottle led by Justin Zook in establishing a truth set for structural variants in the Ashkenazim-Jewish trio data recently released. This work demonstrates the power of this benchmark set – one of the first of its kind for structural variation data – to help understand and refine the accuracies of calling structural variants with a number of approaches.

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June 1, 2021

Alternative splicing in FMR1 premutations carriers

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 sequencing (SMRT) and qRT-PCR we have recently reported that, although the relative abundance of all FMR1 mRNA isoforms is significantly increased in the premutation group compared to controls, there is a disproportionate increase, relative to the overall increase in mRNA, in the abundance of isoforms spliced at both exons 12 and 14. In total, we confirmed the existence of 16 out of 24 predicted isoforms in our samples. However, it is unknown, which isoforms, when overexpressed, may contribute to the premutation pathology. To address this question we have further defined the transcriptional FMR1 isoforms distribution pattern in different tissues, including heart, muscle, brain and testis derived from FXTAS premutation carriers and age-matched controls. Preliminary data indicates the presence of a transcriptional signature of the FMR1 gene, which clusters more by individual than by tissue type. We identified additional isoforms than the 16 reported in our previous study, including a group with particular splice patterns that were observed only in premutations but not in controls. Our findings suggest that the characterization of expression levels of the different FMR1 isoforms is fundamental for understanding the regulation of the FMR1 gene as well as for elucidating the mechanism(s) by which “toxic gain of function” of the FMR1 mRNA may play a role in FXTAS and/or in the other FMR1-associated conditions. In addition to the elevated levels of FMR1 isoforms, the altered abundance/ratio of the corresponding FMRP isomers may affect the overall function of FMRP in premutations.

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June 1, 2021

Effect of coverage depth and haplotype phasing on structural variant detection with PacBio long reads

Each human genome has thousands of structural variants compared to the reference assembly, up to 85% of which are difficult or impossible to detect with Illumina short reads and are only visible with long, multi-kilobase reads. The PacBio RS II and Sequel single molecule, real-time (SMRT) sequencing platforms have made it practical to generate long reads at high throughput. These platforms enable the discovery of structural variants just as short-read platforms did for single nucleotide variants. Numerous software algorithms call structural variants effectively from PacBio long reads, but algorithm sensitivity is lower for insertion variants and all heterozygous variants. Furthermore, the impact of coverage depth and read lengths on sensitivity is not fully characterized. To quantify how zygosity, coverage depth, and read lengths impact the sensitivity of structural variant detection, we obtained high coverage PacBio sequences for three human samples: haploid CHM1, diploid NA12878, and diploid SK-BR-3. For each dataset, reads were randomly subsampled to titrate coverage from 0.5- to 50-fold. The structural variants detected at each coverage were compared to the set at “full” 50-fold coverage. For the diploid samples, additional titrations were performed with reads first partitioned by phase using single nucleotide variants for essentially haploid structural variant discovery. Even at low coverages (1- to 5-fold), PacBio long reads reveal hundreds of structural variants that are not seen in deep 50-fold Illumina whole genome sequences. At moderate 10-fold PacBio coverage, a majority of structural variants are detected. Sensitivity begins to level off at around 40-fold coverage, though it does not fully saturate before 50-fold. Phasing improves sensitivity for all variant types, especially at moderate 10- to 20-fold coverage. Long reads are an effective tool to identify and phase structural variants in the human genome. The majority of variants are detected at moderate 10-fold coverage, and even extremely low long-read coverage (1- to 5-fold) reveals variants that are invisible to short-read sequencing. Performance will continue to improve with better software and longer reads, which will empower studies to connect structural variants to healthy and disease traits in the human population.

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June 1, 2021

The MHC Diversity in Africa Project (MDAP) pilot – 125 African high resolution HLA types from 5 populations

The major histocompatibility complex (MHC), or human leukocyte antigen (HLA) in humans, is a highly diverse gene family with a key role in immune response to disease; and has been implicated in auto-immune disease, cancer, infectious disease susceptibility, and vaccine response. It has clinical importance in the field of solid organ and bone marrow transplantation, where donors and recipient matching of HLA types is key to transplanted organ outcomes. The Sanger based typing (SBT) methods currently used in clinical practice do not capture the full diversity across this region, and require specific reference sequences to deconvolute ambiguity in HLA types. However, reference databases are based largely on European populations, and the full extent of diversity in Africa remains poorly understood. Here, we present the first systematic characterisation of HLA diversity within Africa in the pilot phase of the MHC Diversity in Africa Project, together with an evaluation of methods to carry out scalable cost-effective, as well as reliable, typing of this region in African populations.To sample a geographically representative panel of African populations we obtained 125 samples, 25 each from the Zulu (South Africa), Igbo (Nigeria), Kalenjin (Kenya), Moroccan and Ashanti (Ghana) groups. For methods validation we included two controls from the International Histocompatibility Working Group (IHWG) collection with known typing information. Sanger typing and Illumina HiSeq X sequencing of these samples indicated potentially novel Class I and Class II alleles; however, we found poor correlation between HiSeq X sequencing and SBT for both classes. Long Range PCR and high resolution PacBio RS-II typing of 4 of these samples identified 7 novel Class II alleles, highlighting the high levels of diversity in these populations, and the need for long read sequencing approaches to characterise this comprehensively. We have now expanded this approach to the entire pilot set of 125 samples. We present these confirmed types and discuss a workflow for scaling this to 5000 individuals across Africa.The large number of new alleles identified in our pilot suggests the high level of African HLA diversity and the utility of high resolution methods. The MDAP project will provide a framework for accurate HLA typing, in addition to providing an invaluable resource for imputation in GWAS, boosting power to identify and resolve HLA disease associations.

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June 1, 2021

Target enrichment using a neurology panel for 12 barcoded genomic DNA samples on the PacBio SMRT Sequencing platform

Target enrichment is a powerful tool for studies involved in understanding polymorphic SNPs with phasing, tandem repeats, and structural variations. With increasing availability of reference genomes, researchers can easily design a cost-effective targeted investigation with custom probes specific to regions of interest. Using PacBio long-read technology in conjunction with probe capture, we were able to sequence multi-kilobase enriched regions to fully investigate intronic and exonic regions, distinguish haplotypes, and characterize structural variations. Furthermore, we demonstrate this approach is advantageous for studying complex genomic regions previously inaccessible through other sequencing platforms. In the present work, 12 barcoded genomic DNA (gDNA) samples were sheared to 6 kb for target enrichment analysis using the Neurology panel provided by Roche NimbleGen. Probe-captured DNA was used to make SMRTbell libraries for SMRT Sequencing on the PacBio RS II. Our results demonstrate the ability to multiplex 12 samples and achieve 1300x enrichment of targeted regions. In addition, we achieved an even representation of on-target rate of 70% across the 12 barcoded genomic DNA samples.

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June 1, 2021

“SMRTer Confirmation”: Scalable clinical read-through variant confirmation using the Pacific Biosciences SMRT Sequencing platform

Next-generation sequencing (NGS) has significantly improved the cost and turnaround time for diagnostic genetic tests. ACMG recommends variant confirmation by an orthogonal method, unless sufficiently high sensitivity and specificity can be demonstrated using NGS alone. Most NGS laboratories make extensive use of Sanger sequencing for secondary confirmation of single nucleotide variants (SNVs) and indels, representing a large fraction of the cost and time required to deliver high quality genetic testing data to clinicians and patients. Despite its established data quality, Sanger is not a high-throughput method by today’s standards from either an assay or analysis standpoint as it can involve manual review of Sanger traces and is not amenable to multiplexing. Toward a scalable solution for confirmation, Invitae has developed a fully automated and LIMS-tracked assay and informatics pipeline that utilizes the Pacific Biosciences SMRT sequencing platform. Invitae’s pipeline generates PCR amplicons that encompass the variant(s) of interest, which are converted to closed DNA structures (SMRTbells) and sequenced in pools of 96 per SMRTcell. Each amplicon is appended with a 16nt barcode that encodes the patient and variant IDs. Per-sample de-multiplexing, alignment, variant calling, and confirmation resolution are handled via an automated pipeline. The confirmation process was validated by analyzing 243 clinical SNVs and indels in parallel with the gold standard Sanger sequencing method. Amplicons were sequenced and analyzed in technical replicates to demonstrate reproducibility. In this study, the PacBio-based confirmation pipeline demonstrated high reproducibility (97.5%), and outperformed Sanger in the fraction of primary NGS variants confirmed (PacBio = 93.4% and 94.7% confirmed across two replicates, Sanger = 84.8%) while having 100% concordance of confirmation status among overlapping confirmation calls.

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June 1, 2021

A high-quality genome assembly of SMRT Sequences reveals long-range haplotype structure in the diploid mosquito Aedes aegypti

Aedes aegypti is a tropical and subtropical mosquito vector for Zika, yellow fever, dengue fever, chikungunya, and other diseases. The outbreak of Zika in the Americas, which can cause microcephaly in the fetus of infected women, adds urgency to the need for a high-quality reference genome in order to better understand the organism’s biology and its role in transmitting human disease. We describe the first diploid assembly of an insect genome, using SMRT sequencing and the open-source assembler FALCON-Unzip. This assembly has high contiguity (contig N50 1.3 Mb), is more complete than previous assemblies (Length 1.45 Gb with 87% BUSCO genes complete), and is high quality (mean base >QV30). Long-range haplotype structure, in some cases encompassing more than 4 Mb of extremely divergent homologous sequence, is resolved using a combination of the FALCON-Unzip assembler, genome annotation, coverage depth, and pairwise nucleotide alignments.

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June 1, 2021

Using the PacBio IsoSeq method to search for novel colorectal cancer biomarkers

Early detection of colorectal cancer (CRC) and its precursor lesions (adenomas) is crucial to reduce mortality rates. The fecal immunochemical test (FIT) is a non-invasive CRC screening test that detects the blood-derived protein hemoglobin. However, FIT sensitivity is suboptimal especially in detection of CRC precursor lesions. As adenoma-to-carcinoma progression is accompanied by alternative splicing, tumor-specific proteins derived from alternatively spliced RNA transcripts might serve as candidate biomarkers for CRC detection.

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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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June 1, 2021

Screening and characterization of causative structural variants for bipolar disorder in a significantly linked chromosomal region onXq24-q27 in an extended pedigree from a genetic isolate

Bipolar disorder (BD) is a phenotypically and genetically complex and debilitating neurological disorder that affects 1% of the worldwide population. There is compelling evidence from family, twin and adoption studies supporting the involvement of a genetic predisposition in BD with estimated heritability up to ~ 80%. The risk in first-degree relatives is ten times higher than in the general population. Linkage and association studies have implicated multiple putative chromosomal loci for BP susceptibility, however no disease genes have been identified to date.

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June 1, 2021

Targeted SMRT Sequencing of difficult regions of the genome using a Cas9, non-amplification based method

Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods are reliant upon some form of amplification. Amplification removes the epigenetic marks present in native DNA, and some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, a large number of genetic disorders are caused by expansions of repeat sequences. Furthermore, for some disorders, methylation status has been shown to be a key factor in the mechanism of disease. We have developed a novel, amplification-free enrichment technique that employs the CRISPR/Cas9 system for specific targeting of individual human genes. This method, in conjunction with SMRT Sequencing’s long reads, high consensus accuracy, and uniform coverage, allows the sequencing of complex genomic regions that cannot be investigated with other technologies.

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June 1, 2021

Using the PacBio Sequel System to taxonomically and functionally classify metagenomic samples in a trial of patients undergoing fecal microbiota transplantation

Whole-sample shotgun sequencing can provide a more detailed view of a metagenomic community than 16S sequencing, but its use in multi-sample experiments is limited by throughput, cost and analysis complexity. While short-read sequencing technologies offer higher throughput, read lengthss less fewer than 500 bp will rarely cover a gene of interest, and necessitate assembly before further analysis. Assembling large fragments requires sampling each community member at a high depth, significantly increasing the amount of sequencing needed, and limiting the analysis of rare community members. Assembly methods also risk It is also possible to incorrectly combine combining sequences from different community members.

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June 1, 2021

De novo PacBio long-read assembled avian genomes correct and add to genes important in neuroscience and conservation research

To test the impact of high-quality genome assemblies on biological research, we applied PacBio long-read sequencing in conjunction with the new, diploid-aware FALCON-Unzip assembler to a number of bird species. These included: the zebra finch, for which a consortium-generated, Sanger-based reference exists, to determine how the FALCON-Unzip assembly would compare to the current best references available; Anna’s hummingbird genome, which had been assembled with short-read sequencing methods as part of the Avian Phylogenomics phase I initiative; and two critically endangered bird species (kakapo and ‘alala) of high importance for conservations efforts, whose genomes had not previously been sequenced and assembled.

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June 1, 2021

T-cell receptor profiling using PacBio sequencing of SMARTer libraries

T-cells play a central part in the immune response in humans and related species. T-cell receptors (TCRs), heterodimers located on the T-cell surface, specifically bind foreign antigens displayed on the MHC complex of antigen-presenting cells. The wide spectrum of potential antigens is addressed by the diversity of TCRs created by V(D)J recombination. Profiling this repertoire of TCRs could be useful from, but not limited to, diagnosis, monitoring response to treatments, and examining T-cell development and diversification.

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June 1, 2021

A method for the identification of variants in Alzheimer’s disease candidate genes and transcripts using hybridization capture combined with long-read sequencing

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 causal variants, but are in linkage disequilibrium with the presumed causative variant (Van Cauwenberghe C, et al., The genetic landscape of Alzheimer disease: clinical implications and perspectives. Genet Med 2015;18:421-430).

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