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Saturday, February 20, 2021

Application Note: Microbial multiplexing workflow on the Sequel System

Obtaining microbial genomes with the highest accuracy and contiguity is extremely important when exploring the functional impact of genetic and epigenetic variants on a genome-wide scale. A comprehensive view of the bacterial genome, including genes, regulatory regions, IS elements, phage integration sites, and base modifications is vital to understanding key traits such as antibiotic resistance, virulence, and metabolism. SMRT Sequencing provides complete genomes, often assembled into a single contig. Our streamlined microbial multiplexing procedure for the Sequel System, from library preparation to genome assembly, can be completed with less than 8 hours bench time. Starting with high-quality genomic DNA (gDNA),…

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Saturday, February 20, 2021

Product Note: SMRTbell express template prep 2.0 for microbial multiplexing

The SMRTbell Express Template Prep Kit 2.0 provides a streamlined, single-tube reaction strategy to generate SMRTbell libraries from 500 bp to >50 kb insert size targets to support large-insert genomic libraries, multiplexed microbial genomes and amplicon sequencing. With this new formulation, we have increased both the yield and efficiency of SMRTbell library preparation for SMRT Sequencing while further minimizing handling-induced DNA damage to retain the integrity of genomic DNA (gDNA). This product note highlights the key benefits, performance, and resources available for obtaining complete microbial genome assemblies with multiplexed sequencing. By using a single-tube, addition-only strategy, the streamlined workflow reduces…

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Friday, February 5, 2021

Webinar: Bioinformatics lunch & learn – Better assemblies of bacterial genomes and plasmids with the new microbial assembly pipeline in SMRT Link v8.0

Microbial Assembly is our latest pipeline, specifically designed to assemble bacterial genomes (between 2 and 10 Mb) and plasmids. This pipeline includes the implementation of a new, circular-aware read alignment tool (Raptor), among other algorithmic improvements, which will be covered in this webinar. The topics covered include, staged assembly of bacterial chromosomes and plasmids, implementation of Raptor, a circular-aware read aligner, himeric read detection, origin of replication orientation, troubleshooting and more.

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Tuesday, December 22, 2020

Single chromosomal genome assemblies on the Sequel System with Circulomics high molecular weight DNA extraction for microbes

Background: The Nanobind technology from Circulomics provides an elegant HMW DNA extraction solution for genome sequencing of Gram-positive and -negative microbes. Nanobind is a nanostructured magnetic disk that can be used for rapid extraction of high molecular weight (HMW) DNA from diverse sample types including cultured cells, blood, plant nuclei, and bacteria. Processing can be completed in 7 kb repeats. Fragment size was increased to ~14 kb, with some fragments >30 kb. Results: Here we present a demonstration of these capabilities using isolates relevant to high-throughput sequencing applications, including common foodborne pathogens (Shigella, Listeria, Salmonella), and species often seen in…

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Tuesday, December 22, 2020

Streamlines SMRTbell library generation using addition-only, single tube strategy for all library types reduces time to results

We have streamlined the SMRTbell library generation protocols with improved workflows to deliver seamless end-to-end solutions from sample to analysis. A key improvement is the development of a single-tube reaction strategy that shortened hands-on time needed to generate each SMRTbell library, reduced time-consuming AM Pure purification steps, and minimized sample-handling induced gDNA damage to improve the integrity of long-insert SMRTbell templates for sequencing. The improved protocols support all large-insert genomic libraries, multiplexed microbial genomes, and amplicon sequencing. These advances enable completion of library preparation in less than a day (approximately 4 hours) and opens opportunities for automated library preparation for…

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Tuesday, December 22, 2020

Long Amplicon Analysis: Highly accurate, full-length, phased, allele-resolved gene sequences from multiplexed SMRT Sequencing data.

The correct phasing of genetic variations is a key challenge for many applications of DNA sequencing. Allele-level resolution is strongly preferred for histocompatibility sequencing where recombined genes can exhibit different compatibilities than their parents. In other contexts, gene complementation can provide protection if deleterious mutations are found on only one allele of a gene. These problems are especially pronounced in immunological domains given the high levels of genetic diversity and recombination seen in regions like the Major Histocompatibility Complex. A new tool for analyzing Single Molecule, Real-Time (SMRT) Sequencing data – Long Amplicon Analysis (LAA) – can generate highly accurate,…

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Tuesday, December 22, 2020

Integrative biology of a fungus: Using PacBio SMRT Sequencing to interrogate the genome, epigenome, and transcriptome of Neurospora crassa.

PacBio SMRT Sequencing has the unique ability to directly detect base modifications in addition to the nucleotide sequence of DNA. Because eukaryotes use base modifications to regulate gene expression, the absence or presence of epigenetic events relative to the location of genes is critical to elucidate the function of the modification. Therefore an integrated approach that combines multiple omic-scale assays is necessary to study complex organisms. Here, we present an integrated analysis of three sequencing experiments: 1) DNA sequencing, 2) base-modification detection, and 3) Iso-seq analysis, in Neurospora crassa, a filamentous fungus that has been used to make many landmark…

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Tuesday, December 22, 2020

Assembly of complete KIR haplotypes from a diploid individual by the direct sequencing of full-length fosmids.

We show that linearizing and directly sequencing full-length fosmids simplifies the assembly problem such that it is possible to unambiguously assemble individual haplotypes for the highly repetitive 100-200 kb killer Ig-like receptor (KIR) gene loci of chromosome 19. A tiling of targeted fosmids can be used to clone extended lengths of genomic DNA, 100s of kb in length, but repeat complexity in regions of particular interest, such as the KIR locus, means that sequence assembly of pooled samples into complete haplotypes is difficult and in many cases impossible. The current maximum read length generated by SMRT Sequencing exceeds the length…

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Tuesday, December 22, 2020

Multiplexing human HLA class I & II genotyping with DNA barcode adapters for high throughput research.

Human MHC class I genes HLA-A, -B, -C, and class II genes HLA-DR, -DP and -DQ, play a critical role in the immune system as major factors responsible for organ transplant rejection. The have a direct or linkage-based association with several diseases, including cancer and autoimmune diseases, and are important targets for clinical and drug sensitivity research. HLA genes are also highly polymorphic and their diversity originates from exonic combinations as well as recombination events. A large number of new alleles are expected to be encountered if these genes are sequenced through the UTRs. Thus allele-level resolution is strongly preferred…

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Tuesday, December 22, 2020

Getting the most out of your PacBio libraries with size selection.

PacBio RS II sequencing chemistries provide read lengths beyond 20 kb with high consensus accuracy. The long read lengths of P4-C2 chemistry and demonstrated consensus accuracy of 99.999% are ideal for applications such as de novo assembly, targeted sequencing and isoform sequencing. The recently launched P5-C3 chemistry generates even longer reads with N50 often >10,000 bp, making it the best choice for scaffolding and spanning structural rearrangements. With these chemistry advances, PacBio’s read length performance is now primarily determined by the SMRTbell library itself. Size selection of a high-quality, sheared 20 kb library using the BluePippin™ System has been demonstrated…

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Tuesday, December 22, 2020

SMRT Sequencing solutions for large genomes and transcriptomes.

Single Molecule, Real-Time (SMRT) Sequencing holds promise for addressing new frontiers in large genome complexities, such as long, highly repetitive, low-complexity regions and duplication events, and differentiating between transcript isoforms that are difficult to resolve with short-read technologies. We present solutions available for both reference genome improvement (>100 MB) and transcriptome research to best leverage long reads that have exceeded 20 Kb in length. Benefits for these applications are further realized with consistent use of size-selection of input sample using the BluePippin™ device from Sage Science. Highlights from our genome assembly projects using the latest P5-C3 chemistry on model organisms…

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Tuesday, December 22, 2020

An interactive workflow for the analysis of contigs from the metagenomic shotgun assembly of SMRT Sequencing data.

The data throughput of next-generation sequencing allows whole microbial communities to be analyzed using a shotgun sequencing approach. Because a key task in taking advantage of these data is the ability to cluster reads that belong to the same member in a community, single-molecule long reads of up to 30 kb from SMRT Sequencing provide a unique capability in identifying those relationships and pave the way towards finished assemblies of community members. Long reads become even more valuable as samples get more complex with lower intra-species variation, a larger number of closely related species, or high intra-species variation. Here we…

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Tuesday, December 22, 2020

A comparison of assemblers and strategies for complex, large-genome sequencing with PacBio long reads.

PacBio sequencing holds promise for addressing large-genome complexities, such as long, highly repetitive, low-complexity regions and duplication events that are difficult to resolve with short-read technologies. Several strategies, with varying outcomes, are available for de novo sequencing and assembling of larger genomes. Using a diploid fungal genome, estimated to be ~80 Mb in size, as the basis dataset for comparison, we highlight assembly options when using only PacBio sequencing or a combined strategy leveraging data sets from multiple sequencing technologies. Data generated from SMRT Sequencing was subjected to assembly using different large-genome assemblers, and comparisons of the results will be…

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Tuesday, December 22, 2020

A workflow for the analysis of contigs from the metagenomic shotgun assembly of SMRT Sequencing data

The throughput of SMRT Sequencing and long reads allows microbial communities to be analyzed using a shotgun sequencing approach. Key to leveraging this data is the ability to cluster sequences belonging to the same member of a community. Long reads of up to 40 kb provide a unique capability in identifying those relationships, and pave the way towards finished assemblies of community members. Long reads are highly valuable when samples are more complex and containing lower intra-species variation, such as a larger number of closely related species, or high intra-species variation. Here, we present a collection of tools tailored for…

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Tuesday, December 22, 2020

Resources for advanced bioinformaticians working in plant and animal genomes with SMRT Sequencing.

Significant advances in bioinformatics tool development have been made to more efficiently leverage and deliver high-quality genome assemblies with PacBio long-read data. Current data throughput of SMRT Sequencing delivers average read lengths ranging from 10-15 kb with the longest reads exceeding 40 kb. This has resulted in consistent demonstration of a minimum 10-fold improvement in genome assemblies with contig N50 in the megabase range compared to assemblies generated using only short- read technologies. This poster highlights recent advances and resources available for advanced bioinformaticians and developers interested in the current state-of-the-art large genome solutions available as open-source code from PacBio…

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