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Asset Tag: HiFi sequencing

June 1, 2021

Low-input single molecule HiFi sequencing for metagenomic samples

HiFi sequencing on the PacBio Sequel II System enables complete microbial community profiling of complex metagenomic samples using whole genome shotgun sequences. With HiFi sequencing, highly accurate long reads overcome the challenges posed by the presence of intergenic and extragenic repeat elements in microbial genomes, thus greatly improving phylogenetic profiling and sequence assembly. Recent improvements in library construction protocols enable HiFi sequencing starting from as low as 5 ng of input DNA. Here, we demonstrate comparative analyses of a control sample of known composition and a human fecal sample from varying amounts of input genomic DNA (1 ug, 200 ng, 5 ng), and present the corresponding library preparation workflows for standard, low input, and Ultra-Low methods. We demonstrate that the metagenome assembly, taxonomic assignment, and gene finding analyses are comparable across all methods for both samples, providing access to HiFi sequencing even for DNA-limited sample types.

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June 1, 2021

Metagenomic analysis of type II diabetes gut microbiota using PacBio HiFi reads reveals taxonomic and functional differences

In the past decade, the human microbiome has been increasingly shown to play a major role in health. For example, imbalances in gut microbiota appear to be associated with Type II diabetes mellitus (T2DM) and cardiovascular disease. Coronary artery disease (CAD) is a major determinant of the long-term prognosis among T2DM patients, with a 2- to 4-fold increased mortality risk when present. However, the exact microbial strains or functions implicated in disease need further investigation. From a large study with 523 participants (185 healthy controls, 186 T2DM patients without CAD, and 106 T2DM patients with CAD), 3 samples from each patient group were selected for long read sequencing. Each sample was prepared and sequenced on one Sequel II System SMRT Cell, to assess whether long accurate PacBio HiFi reads could yield additional insights to those made using short reads. Each of the 9 samples was subject to metagenomic assembly and binning, taxonomic classification and functional profiling. Results from metagenomic assembly and binning show that it is possible to generate a significant number of complete MAGs (Metagenome Assembled Genomes) from each sample, with over half of the high-quality MAGs being represented by a single circular contig. We show that differences found in taxonomic and functional profiles of healthy versus diabetic patients in the small 9-sample study align with the results of the larger study, as well as with results reported in literature. For example, the abundances of beneficial short- chain fatty acid (SCFA) producers such as Phascolarctobacterium faecium and Faecalibacterium prausnitzii were decreased in T2DM gut microbiota in both studies, while the abundances of quinol and quinone biosynthesis pathways were increased as compared to healthy controls. In conclusion, metagenomic analysis of long accurate HiFi reads revealed important taxonomic and functional differences in T2DM versus healthy gut microbiota. Furthermore, metagenome assembly of long HiFi reads led to the recovery of many complete MAGs and a significant number of complete circular bacterial chromosome sequences.

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June 1, 2021

Comprehensive variant detection in a human genome with highly accurate long reads

Introduction: Long-read sequencing has revealed more than 20,000 structural variants spanning over 12 Mb in a healthy human genome. Short-read sequencing fails to detect most structural variants but has remained the more effective approach for small variants, due to 10-15% error rates in long reads, and copy-number variants (CNVs), due to lack of effective long-read variant callers. The development of PacBio highly accurate long reads (HiFi reads) with read lengths of 10-25 kb and quality >99% presents the opportunity to capture all classes of variation with one approach.Methods: We sequence the Genome in a Bottle benchmark sample HG002 and an individual with a presumed Mendelian disease with HiFi reads. We call SNVs and indels with DeepVariant and extend the structural variant caller pbsv to call CNVs using read depth and clipping signatures. Results: For 18-fold coverage with 13 kb HiFi reads, variant calling in HG002 achieves an F1 score of 99.7% for SNVs, 96.6% for indels, and 96.4% for structural variants. Additionally, we detect more than 300 CNVs spanning around 10 Mb. For the Mendelian disease case, HiFi reads reveal thousands of variants that were overlooked by short-read sequencing, including a candidate causative structural variant. Conclusions: These results illustrate the ability of HiFi reads to comprehensively detect variants, including those associated with human disease.

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June 1, 2021

Full-Length Sequencing of CYP2D6Variants with PacBio HiFi Reads

CYP2D6 is a highly polymorphic gene with more than 130 named variants, including deletions, duplications, single nucleotide polymorphisms, and other types of variation (Butler, 2018; Black et al., 2011). These variants affect the rate of metabolism in human individuals of approximately 25% of common prescription drugs (Owen et al., 2019;). PacBio SMRT sequencing is a proven tool for the interrogation of CYP2D6 variants (Qiao et al., 2016; Buermans et al., 2017).  Now with HiFi sequencing, we have developed a streamlined end-to-end workflow for the more accurate detection of highly polymorphic CYP2D6 loci. This study also evaluates the advantage of HiFi reads for the sequencing of full-length CYP2D6 genes with variants previously annotated by other technologies.

Twenty-two Coriell pharmacogenomic samples containing variant CYP2D6 alleles were amplified using long-range PCR. The primer pairs for the amplification of upstream CYP2D6 gene duplications and the downstream CYP2D6 genes were adapted from a publication in Pharmacogenomics (Qiao et al., 2019). A 2-step PCR method was used for the addition of the unique barcode to each sample, allowing pooling of multiple samples for SMRTbell library prep. The resulting SMRTbell Library was then sequenced on the PacBio Sequel II/IIe system for 20-hours. HiFi reads (>QV20) were demultiplexed on SMRTlink and clustered into haplotypes. The consensus reads of each haplotype were produced using the “pbaa” amplicon analysis and then mapped to the human reference genome GRCh38 for the assignment of CYP2D6 types.

More than 700,000 full-length HiFi reads were generated with an average read length of 8.2 kb and a mean accuracy of 99.9%. Nearly all (>99%) demultiplexed reads were on target to the CYP2D6 locus. Genotyping of the CYP2D6 region with PacBio HiFi reads identified all expected upstream duplications and downstream CYP2D6 alleles including single nucleotide variants, except for *5 allele which is a complete deletion. For 21 of 22 samples, the types from HiFi reads matched the diplotypes identified from microarrays and qPCR, while providing full resolution of each allele. One sample was identified as being mistyped by microarray as *1/*41. HiFi sequencing produced a correct type of *33/*41. In addition, for 4/21 samples HiFi sequencing identified duplications missed by microarray or real-time PCR.

The PCR and sequencing assay we have presented here for the detection of CYP2D6 variants is robust and specific. Assignment of new alleles or duplications on pharmacogenomic samples from HiFi reads suggests that PacBio sequencing technology can reveal new diplotypes that were not characterized accurately by other technologies. This study demonstrates that HiFi sequencing provides much higher resolution than either microarray or real-time PCR for the detection of polymorphic genes, while maintaining sensitivity and accuracy.

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