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June 1, 2021  |  

Immune regions are no longer incomprehensible with SMRT Sequencing

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 efforts, the fine mapping of causal variants of immune genes for their well-documented association with cancer, drug-induced hypersensitivity and immune-related diseases, has been slower than expected. This has in many ways limited our understanding of the mechanisms leading to immune disease. In the present work, we demonstrate the advantages of long reads delivered by SMRT Sequencing for assembling complete haplotypes of MHC and KIR gene clusters, as well as calling correct genotypes of genes comprised within them. All the genotype information is detected at allele- level with full phasing information across SNP-poor regions. Genotypes were called correctly from targeted gene amplicons, haplotypes, as well as from a completely assembled 5 Mb contig of the MHC region from a de novo assembly of whole genome shotgun data. De novo analysis pipeline used in all these approaches allowed for reference-free analysis without imputation, a key for interrogation without prior knowledge about ethnic backgrounds. These methods are thus easily adoptable for previously uncharacterized human or non-human species.

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June 1, 2021  |  

An improved circular consensus algorithm with an application to detection of HIV-1 Drug-Resistance Associated Mutations (DRAMs)

Scientists who require confident resolution of heterogeneous populations across complex regions have been unable to transition to short-read sequencing methods. They continue to depend on Sanger Sequencing despite its cost and time inefficiencies. Here we present a new redesigned algorithm that allows the generation of circular consensus sequences (CCS) from individual SMRT Sequencing reads. With this new algorithm, dubbed CCS2, it is possible to reach arbitrarily high quality across longer insert lengths at a lower cost and higher throughput than Sanger Sequencing. We apply this new algorithm, dubbed CCS2, to the characterization of the HIV-1 K103N drug-resistance associated mutation, which is both important clinically, and represents a challenge due to regional sequence context. A mutation was introduced into the 3rd position of amino acid position 103 (A>C substitution) of the RT gene on a pNL4-3 backbone by site-directed mutagenesis. Regions spanning ~1,300 bp were PCR amplified from both the non-mutated and mutant (K103N) plasmids, and were sequenced individually and as a 50:50 mixture. Sequencing data were analyzed using the new CCS2 algorithm, which uses a fully-generative probabilistic model of our SMRT Sequencing process to polish consensus sequences to arbitrarily high accuracy. This result, previously demonstrated for multi-molecule consensus sequences with the Quiver algorithm, is made possible by incorporating per-Zero Mode Waveguide (ZMW) characteristics, thus accounting for the intrinsic changes in the sequencing process that are unique to each ZMW. With CCS2, we are able to achieve a per-read empirical quality of QV30 with 19X coverage. This yields ~5000 1.3 kb consensus sequences with a collective empirical quality of ~QV40. Additionally, we demonstrate a 0% miscall rate in both unmixed samples, and estimate a 48:52% frequency for the K103N mutation in the mixed sample, consistent with data produced by orthogonal platforms.

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June 1, 2021  |  

Targeted sequencing and chromosomal haplotype assembly using TLA and SMRT Sequencing

With the increasing availability of whole-genome sequencing, haplotype reconstruction of individual genomes, or haplotype assembly, remains unsolved. Like the de novo genome assembly problem, haplotype assembly is greatly simplified by having more long-range information. The Targeted Locus Amplification (TLA) technology from Cergentis has the unique capability of targeting a specific region of the genome using a single primer pair and yielding ~2 kb DNA circles that are comprised of ~500 bp fragments. Fragments from the same circle come from the same haplotype and follow an exponential decay in distance from the target region, with a span that reaches the multi-megabase range. Here, we apply TLA to the BRCA1 gene on NA12878 and then sequence the resulting 2 kb circles on a PacBio RS II. The multiple fragments per circle were iteratively mapped to hg19 and then haplotype assembled using HAPCUT. We show that the 80 kb length of BRCA1 is represented by a single haplotype block, which was validated against GIAB data. We then explored chromosomal-scale haplotype assembly by combining these data with whole genome shotgun PacBio long reads, and demonstrate haplotype blocks approaching the length of chromosome 17 on which BRCA1 lies. Finally, by performing TLA without the amplification step and size selecting for reads >5 kb to maximize the number of fragments per read, we target whole genome haplotype assembly across all chromosomes.

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June 1, 2021  |  

Highly sensitive and cost-effective detection of somatic cancer variants using single-molecule, real-time sequencing

Next-Generation Sequencing (NGS) technologies allow for molecular profiling of cancer samples with high sensitivity and speed at reduced cost. For efficient profiling of cancer samples, it is important that the NGS methods used are not only robust, but capable of accurately detecting low-frequency somatic mutations. Single Molecule, Real-Time (SMRT) Sequencing offers several advantages, including the ability to sequence single molecules with very high accuracy (>QV40) using the circular consensus sequencing (CCS) approach. The availability of genetically defined, human genomic reference standards provides an industry standard for the development and quality control of molecular assays for studying cancer variants. Here we characterize SMRT Sequencing for the detection of low-frequency somatic variants using the Quantitative Multiplex DNA Reference Standards from Horizon Discovery, combined with amplification of the variants using the Multiplicom Tumor Hotspot MASTR Plus assay. First, we sequenced a reference standard containing precise allelic frequencies from 1% to 24.5% for major oncology targets verified using digital PCR. This reference material recapitulates the complexity of tumor composition and serves as a well-characterized control. The control sample was amplified using the Multiplicom Tumor Hotspot MASTR Plus assay that targets 252 amplicons (121-254 bp) from 26 relevant cancer genes, which includes all 11 variants in the control sample. Next, we sequenced control samples prepared by SeraCare Life Sciences, which contained a defined mutation at allelic frequencies from 10% down to 0.1%. The wild type and mutant amplicons were serially diluted, sequenced and analyzed using SMRT Sequencing to identify the variants and determine the observed frequency. The random error profile and high-accuracy CCS reads make it possible to accurately detect low-frequency somatic variants.

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June 1, 2021  |  

Candidate gene screening using long-read sequencing

We have developed several candidate gene screening applications for both Neuromuscular and Neurological disorders. The power behind these applications comes from the use of long-read sequencing. It allows us to access previously unresolvable and even unsequencable genomic regions. SMRT Sequencing offers uniform coverage, a lack of sequence context bias, and very high accuracy. In addition, it is also possible to directly detect epigenetic signatures and characterize full-length gene transcripts through assembly-free isoform sequencing. In addition to calling the bases, SMRT Sequencing uses the kinetic information from each nucleotide to distinguish between modified and native bases.

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June 1, 2021  |  

Application-specific barcoding strategies for SMRT Sequencing

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.

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June 1, 2021  |  

Highly contiguous de novo human genome assembly and long-range haplotype phasing using SMRT Sequencing

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.

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June 1, 2021  |  

An improved circular consensus algorithm with an application to detect HIV-1 Drug Resistance Associated Mutations (DRAMs)

Scientists who require confident resolution of heterogeneous populations across complex regions have been unable to transition to short-read sequencing methods. They continue to depend on Sanger sequencing despite its cost and time inefficiencies. Here we present a new redesigned algorithm that allows the generation of circular consensus sequences (CCS) from individual SMRT Sequencing reads. With this new algorithm, dubbed CCS2, it is possible to reach high quality across longer insert lengths at a lower cost and higher throughput than Sanger sequencing. We applied CCS2 to the characterization of the HIV-1 K103N drug-resistance associated mutation in both clonal and patient samples. This particular DRAM has previously proved to be clinically relevant, but challenging to characterize due to regional sequence context. First, a mutation was introduced into the 3rd position of amino acid position 103 (A>C substitution) of the RT gene on a pNL4-3 backbone by site-directed mutagenesis. Regions spanning ~1.3 kb were PCR amplified from both the non-mutated and mutant (K103N) plasmids, and were sequenced individually and as a 50:50 mixture. Additionally, the proviral reservoir of a subject with known dates of virologic failure of an Efavirenz-based regimen and with documented emergence of drug resistant (K103N) viremia was sequenced at several time points as a proof-of-concept study to determine the kinetics of retention and decay of K103N.Sequencing data were analyzed using the new CCS2 algorithm, which uses a fully-generative probabilistic model of our SMRT Sequencing process to polish consensus sequences to high accuracy. With CCS2, we are able to achieve a per-read empirical quality of QV30 (99.9% accuracy) at 19X coverage. A total of ~5000 1.3 kb consensus sequences with a collective empirical quality of ~QV40 (99.99%) were obtained for each sample. We demonstrate a 0% miscall rate in both unmixed control samples, and estimate a 48:52 frequency for the K103N mutation in the mixed (50:50) plasmid sample, consistent with data produced by orthogonal platforms. Additionally, the K103N escape variant was only detected in proviral samples from time points subsequent (19%) to the emergence of drug resistant viremia. This tool might be used to monitor the HIV reservoir for stable evolutionary changes throughout infection.

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June 1, 2021  |  

Complete telomere-to-telomere de novo assembly of the Plasmodium falciparum genome using long-read sequencing

Sequence-based estimation of genetic diversity of Plasmodium falciparum, the most lethal malarial parasite, has proved challenging due to a lack of a complete genomic assembly. The skewed AT-richness (~80.6% (A+T)) of its genome and the lack of technology to assemble highly polymorphic sub-telomeric regions that contain clonally variant, multigene virulence families (i.e. var and rifin) have confounded attempts using short-read NGS technologies. Using single molecule, real-time (SMRT) sequencing, we successfully compiled all 14 nuclear chromosomes of the P. falciparum genome from telomere-to-telomere in single contigs. Specifically, amplification-free sequencing generated reads of average length 12 kb, with =50% of the reads between 15.5 and 50 kb in length. A hierarchical genome assembly process (HGAP), was used to assemble the P. falciparum genome de novo. This assembly accurately resolved centromeres (~90-99% (A+T)) and sub-telomeric regions, and identified large insertions and duplications in the genome that added extra genes to the var and rifin virulence families, along with smaller structural variants such as homopolymer tract expansions. These regions can be used as markers for genetic diversity during comparative genome analyses. Moreover, identifying the polymorphic and repetitive sub-telomeric sequences of parasite populations from endemic areas might inform the link between structural variation and phenotypes such as virulence, drug resistance and disease transmission.

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June 1, 2021  |  

Whole gene sequencing of KIR-3DL1 with SMRT Sequencing and the distribution of allelic variants in different ethnic groups

The killer-cell immunoglobulin-like receptor (KIR) gene family are involved in immune modulation during viral infection, autoimmune disease and in allogeneic stem cell transplantation. Most KIR gene diversity studies and their impact on the transplant outcome is performed by gene absence/presence assays. However, it is well known that KIR gene allelic variations have biological significance. Allele level typing of KIR genes has been very challenging until recently due to the homologous nature of those genes and very long intronic sequences. SMRT (Single Molecule Real-Time) Sequencing generates average long reads of 10 to 15 kb and allows us to obtain in-phase long sequence reads. We have developed a PCR assay for SMRT Sequencing on the PacBio RS II platform in our lab for 3DL1 whole gene sequencing. This approach allows us to obtain allele level typing for 3DL1 genes and could serve as a model to type other KIR genes at allelic level.

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June 1, 2021  |  

Multiplex target enrichment using barcoded multi-kilobase fragments and probe-based capture technologies

Target enrichment capture methods allow scientists to rapidly interrogate important genomic regions of interest for variant discovery, including SNPs, gene isoforms, and structural variation. Custom targeted sequencing panels are important for characterizing heterogeneous, complex diseases and uncovering the genetic basis of inherited traits with more uniform coverage when compared to PCR-based strategies. With the increasing availability of high-quality reference genomes, customized gene panels are readily designed with high specificity to capture genomic regions of interest, thus enabling scientists to expand their research scope from a single individual to larger cohort studies or population-wide investigations. Coupled with PacBio® long-read sequencing, these technologies can capture 5 kb fragments of genomic DNA (gDNA), which are useful for interrogating intronic, exonic, and regulatory regions, characterizing complex structural variations, distinguishing between gene duplications and pseudogenes, and interpreting variant haplotyes. In addition, SMRT® Sequencing offers the lowest GC-bias and can sequence through repetitive regions. We demonstrate the additional insights possible by using in-depth long read capture sequencing for key immunology, drug metabolizing, and disease causing genes such as HLA, filaggrin, and cancer associated genes.

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June 1, 2021  |  

Collection of major HLA allele sequences in Japanese population toward the precise NGS based HLA DNA typing at the field 4 level

We previously reported on the use of the Ion PGM next generation sequencing (NGS) platform to genotype HLA class I and class II genes by a super-high resolution, single-molecule, sequence-based typing (SS-SBT) method (Shiina et al. 2012). However, HLA alleles could not be assigned at the field 4 level at some HLA loci such as DQA1, DPA1 and DPB1 because the SNP and indel densities were too low to identify and separate both of the phases. In this regard, we have now added the single molecule, real-time (SMRT) DNA sequencer PacBio RS II method to our analysis in order to test whether it might determine the HLA allele sequences in some of the loci with which we previously had difficulties. In this study, we report on sequence-based genotyping of entire HLA gene sequences from the promoter-enhancer region to 3’UTR of the major HLA loci (A, B, C, DRB1, DRB345, DQA1, DQB1, DPA1 and DPB1) using 46 Japanese reference subjects who represented a distribution of more than 99.5% of the HLA alleles at each of the HLA loci and the PacBio RS II and Ion PGM systems.

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June 1, 2021  |  

Analysis of 37,000 Caucasian samples reveals tight linkage between SNP RS9277534 and high resolution typing of HLA-DPB1

HLA-DPB1 mismatching between patients and unrelated donors is known to increase the risk of acute graft-versus-host-disease (GvHD) after hematopoietic stem cell transplantation. If only HLA-DPB1 mismatched donors are available, the genotype defined by the Single Nucleotide Polymorphism (SNP) rs9277534 can be used to select mismatched donors that are well-tolerated. However, since rs9277534 resides within the 3’ untranslated region (UTR), it usually is not analyzed during DPB1 routine typing.

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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 risk 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). Long-read sequencing together with hybrid-capture targeting technologies provides a powerful combination to target candidate genes/transcripts of interest. Shearing the genomic DNA to ~5 kb fragments and then capturing with probes that span the whole gene(s) of interest can provide uniform coverage across the entire region, identifying variants and allowing for phasing into two haplotypes. Furthermore, capturing full-length cDNA from the same sample using the same capture probes can also provide an understanding of isoforms that are generated and allow them to be assigned to their corresponding haplotype. Here we present a method for capturing genomic DNA and cDNA from an AD sample using a panel of probes targeting approximately 20 late-onset AD candidate genes which includes CLU, ABCA7, CD33, TREM2, TOMM40, PSEN2, APH1 and BIN1. By combining xGen® Lockdown® probes with SMRT Sequencing, we provide completely sequenced candidate genes as well as their corresponding transcripts. In addition, we are also able to evaluate structural variants that due to their size, repetitive nature, or low sequence complexity have been un-sequenceable using short-read technologies.

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June 1, 2021  |  

Characterization of the Poly-T variants in the TOMM40 gene using PacBio long reads

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 reads not only provide continuous sequence data though polymorphic or repetitive regions, but also have no GC bias. Here we describe the characterization of the poly-T locus in TOMM40, a gene known to be associated with progression to Alzheimer’s, using PacBio long reads. Probes were designed to capture a 20 kb region comprising the TOMM40 and ApoE genes. Target regions were captured in multiple cell lines and sequencing libraries made using standard sample preparation methods. We will present our results on the poly-T structural variants that we observed in TOMM40 in these cell lines. We will also present our results on probe design optimization and barcoding strategies for a cost-effective solution.

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