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Tuesday, June 1, 2021

Profiling complex population genomes with highly accurate single molecule reads: cow rumen microbiomes

Determining compositions and functional capabilities of complex populations is often challenging, especially for sequencing technologies with short reads that do not uniquely identify organisms or genes. Long-read sequencing improves the resolution of these mixed communities, but adoption for this application has been limited due to concerns about throughput, cost and accuracy. The recently introduced PacBio Sequel System generates hundreds of thousands of long and highly accurate single-molecule reads per SMRT Cell. We investigated how the Sequel System might increase understanding of metagenomic communities. In the past, focus was largely on taxonomic classification with 16S rRNA sequencing. Recent expansion to WGS…

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Tuesday, 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…

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Tuesday, June 1, 2021

De novo assembly and preliminary annotation of the Schizocardium californicum genome

Animals in the phylum Hemichordata have provided key understanding of the origins and development of body patterning and nervous system organization. However, efforts to sequence and assemble the genomes of highly heterozygous non-model organisms have proven to be difficult with traditional short read approaches. Long repetitive DNA structures, extensive structural variation between haplotypes in polyploid species, and large genome sizes are limiting factors to achieving highly contiguous genome assemblies. Here we present the highly contiguous de novo assembly and preliminary annotation of an indirect developing hemichordate genome, Schizocardium californicum, using SMRT Sequening long reads.

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Tuesday, June 1, 2021

Best practices for whole genome sequencing using the Sequel System

Plant and animal whole genome sequencing has proven to be challenging, particularly due to genome size, high density of repetitive elements and heterozygosity. The Sequel System delivers long reads, high consensus accuracy and uniform coverage, enabling more complete, accurate, and contiguous assemblies of these large complex genomes. The latest Sequel chemistry increases yield up to 8 Gb per SMRT Cell for long insert libraries >20 kb and up to 10 Gb per SMRT Cell for libraries >40 kb. In addition, the recently released SMRTbell Express Template Prep Kit reduces the time (~3 hours) and DNA input (~3 µg), making the…

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Tuesday, June 1, 2021

Haplotyping using full-length transcript sequencing reveals allele-specific expression

An important need in analyzing complex genomes is the ability to separate and phase haplotypes. While whole genome assembly can deliver this information, it cannot reveal whether there is allele-specific gene or isoform expression. The PacBio Iso-Seq method, which can produce high-quality transcript sequences of 10 kb and longer, has been used to annotate many important plant and animal genomes. We present an algorithm called IsoPhase that post-processes Iso-Seq data for transcript-based haplotyping. We applied IsoPhase to a maize Iso-Seq dataset consisting of two homozygous parents and two F1 cross hybrids. We validated the majority of the SNPs called with…

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Tuesday, June 1, 2021

A low DNA input protocol for high-quality PacBio de novo genome assemblies from single invertebrate individuals

A high-quality reference genome is an essential tool for studies of plant and animal genomics. PacBio Single Molecule, Real-Time (SMRT) Sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. PacBio is the core technology for many large genome initiatives, however, relatively high DNA input requirements (5 µg for standard library protocol) have placed PacBio out of reach for many projects on small, non-inbred organisms that may have lower DNA content. Here we present high-quality de novo genome assemblies from single invertebrate individuals for two different species: the Anopheles…

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Tuesday, June 1, 2021

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, June 1, 2021

Single molecule high-fidelity (HiFi) Sequencing with >10 kb libraries

Recent improvements in sequencing chemistry and instrument performance combine to create a new PacBio data type, Single Molecule High-Fidelity reads (HiFi reads). Increased read length and improvement in library construction enables average read lengths of 10-20 kb with average sequence identity greater than 99% from raw single molecule reads. The resulting reads have the accuracy comparable to short read NGS but with 50-100 times longer read length. Here we benchmark the performance of this data type by sequencing and genotyping the Genome in a Bottle (GIAB) HG0002 human reference sample from the National Institute of Standards and Technology (NIST). We…

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Tuesday, June 1, 2021

A complete solution for high-quality genome annotation using the PacBio Iso-Seq method

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. We describe here the full Iso-Seq ecosystem that enables researchers to achieve high-quality genome annotations. The Iso-Seq Express workflow is a 1-day protocol that requires only 60-300 ng of total RNA and supports multiplexing of different tissues. Sequencing on a single SMRT Cell 8M on the Sequel II System produces up to 4 million full-length reads, sufficient to exhaustively characterize a whole transcriptome on the order of 15,000-17,000 genes with 100,000 or more…

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Friday, February 5, 2021

PAG Conference: Update on sequencing of the Cabernet sauvignon genome

Grant Cramer from the University of Nevada, Reno, and Dario Cantu from the Univeristy of Callifornia, Davis, discuss past challenges with sequencing Clone 8 of Cabernet Sauvignon (Vitis vinifera). An assembly of the genome was attempted with approximately 110x Illumina reads and 5x PacBio reads. The PacBio SMRT Sequencing read made major improvements in the assembly compared with the results of Illumina reads only. However, the assembly results were still unsatisfactory, so an additional 100-fold SMRT Sequencing coverage had been generated. An update on the current sequencing results and status of the assembly are presented.

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Friday, February 5, 2021

PAG 2016 Highlights: Customer interviews

See what PacBio users had to say about SMRT Sequencing at the Plant and Animal Genome (PAG) Conference in San Diego. This brief video captures highlights from posters, presentations, and the exhibit hall. See how SMRT Sequencing benefits plant and animal scientists in their genomic investigations. [Engligh, some Mandarin]

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Friday, February 5, 2021

PAG PacBio Workshop: Introducing 5 new high-quality PacBio genome assemblies for rice to help solve the 10-billion people question

At PAG 2017, Rod Wing presented five new, high-quality rice genome assemblies developed with SMRT Sequencing, including one that has eight complete chromosomes including centromeres. He also offered an early look at data generated with the Sequel System for a new assembly underway. This work is done with the goal of developing rice varieties that will be better suited to feeding a rapidly growing global population.

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Friday, February 5, 2021

PAG PacBio Workshop: Genome assembly and molecular genetics of the dengue, yellow fever, and zika vector Aedes aegypti

In this PAG 2017 presentation, Ben Matthews describes a new genome assembly for Aedes aegypti, the mosquito responsible for spreading Zika virus, yellow fever, and other infectious diseases. By using PacBio long-read sequencing, scientists produced an assembly that is much more complete and contiguous than a previous assembly; 7,500 transcripts map to the new contigs but not to the old assembly. The genome is important for designing guide RNAs for CRISPR, understanding resistance to mosquito repellants, and much more.

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