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Tuesday, December 22, 2020

Library prep and bioinformatics improvements for full-length transcript sequencing on the PacBio Sequel System

The PacBio Iso-Seq method produces high-quality, full-length transcripts of up to 10 kb and longer and has been used to annotate many important plant and animal genomes. Here we describe an improved, simplified library workflow and analysis pipeline that reduces library preparation time, RNA input, and cost. The Iso-Seq V2 Express workflow is a one day protocol that requires only ~300 ng of total RNA input while also reducing the number of reverse transcription and amplification steps down to single reactions. Compared with the previous workflow, the Iso-Seq V2 Express workflow increases the percentage of full-length (FL) reads while achieving…

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Tuesday, December 22, 2020

Single cell isoform sequencing (scIso-Seq) identifies novel full-length mRNAs and cell type-specific expression

Single cell RNA-seq (scRNA-seq) is an emerging field for characterizing cell heterogeneity in complex tissues. However, most scRNA-seq methodologies are limited to gene count information due to short read lengths. Here, we combine the microfluidics scRNA-seq technique, Drop-Seq, with PacBio Single Molecule, Real-Time (SMRT) Sequencing to generate full-length transcript isoforms that can be confidently assigned to individual cells. We generated single cell Iso-Seq (scIso-Seq) libraries for chimp and human cerebral organoid samples on the Dolomite Nadia platform and sequenced each library with two SMRT Cells 8M on the PacBio Sequel II System. We developed a bioinformatics pipeline to identify, classify,…

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Tuesday, December 22, 2020

Improving long-read assembly of microbial genomes and plasmids

Complete, high-quality microbial genomes are very valuable across a broad array of fields, from environmental studies, to human microbiome health, food pathogen surveillance, etc. Long-read sequencing enables accurate resolution of complex microbial genomes and is becoming the new standard. Here we report our novel Microbial Assembly pipeline to facilitate rapid, large-scale analysis of microbial genomes. We sequenced a 48-plex library with one SMRT Cell 8M on the Sequel II System, demultiplexed, then analyzed the data with Microbial Assembly.

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Tuesday, December 22, 2020

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…

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Tuesday, December 22, 2020

Using whole exome sequencing and bacterial pathogen sequencing to investigate the genetic basis of pulmonary non-tuberculous mycobacterial infections.

Pulmonary non-tuberculous mycobacterial (PNTM) infections occur in patients with chronic lung disease, but also in a distinct group of elderly women without lung defects who share a common body morphology: tall and lean with scoliosis, pectus excavatum, and mitral valve prolapse. In order to characterize the human host susceptibility to PNTM, we performed whole exome sequencing (WES) of 44 individuals in extended families of patients with active PNTM as well as 55 additional unrelated individuals with PNTM. This unique collection of familial cohorts in PNTM represents an important opportunity for a high yield search for genes that regulate mucosal immunity.…

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Tuesday, December 22, 2020

Highly sensitive, non-invasive detection of colorectal cancer mutations using single molecule, third generation sequencing.

Colorectal cancer (CRC) represents one of the most prevalent and lethal malignant neoplasms and every individual of age 50 and above should undergo regular CRC screening. Currently, the most effective procedure to detect adenomas, the precursors to CRC, is colonoscopy, which reduces CRC incidence by 80%. However, it is an invasive approach that is unpleasant for the patient, expensive, and poses some risk of complications such as colon perforation. A non-invasive screening approach with detection rates comparable to those of colonoscopy has not yet been established. The current study applies Pacific Biosciences third generation, single molecule sequencing to the inspection…

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Tuesday, December 22, 2020

Evaluation of multiplexing strategies for HLA genotyping using PacBio Sequencing technology.

Fully phased allele-level sequencing of highly polymorphic HLA genes is greatly facilitated by SMRT Sequencing technology. In the present work, we have evaluated multiple DNA barcoding strategies for multiplexing several loci from multiple individuals, using three different tagging methods. Specifically MHC class I genes HLA-A, -B, and –C were indexed via DNA Barcodes by either tailed primers or barcoded SMRTbell adapters. Eight different 16-bp barcode sequences were used in symmetric & asymmetric pairing. Eight DNA barcoded adapters in symmetric pairing were independently ligated to a pool of HLA-A, -B and –C for eight different individuals, one at a time and…

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Tuesday, December 22, 2020

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…

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Tuesday, December 22, 2020

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…

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Tuesday, December 22, 2020

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…

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Tuesday, December 22, 2020

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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Tuesday, December 22, 2020

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…

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Tuesday, December 22, 2020

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…

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Tuesday, December 22, 2020

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…

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Tuesday, December 22, 2020

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…

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