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Monday, October 21, 2019

SMRT Sequencing of DNA and RNA samples extracted from formalin-fixed and paraffin embedded tissues using adaptive focused acoustics by Covaris.

Recent advances in next-generation sequencing have led to an increased use of formalin-fixed and paraffin-embedded (FFPE) tissues for medical samples in disease and scientific research. Single Molecule, Real-Time (SMRT) Sequencing offers a unique advantage for direct analysis of FFPE samples without amplification. However, obtaining ample long-read information from FFPE samples has been a challenge due to the quality and quantity of the extracted DNA. FFPE samples often contain damaged sites, including breaks in the backbone and missing or altered nucleotide bases, which directly impact sequencing and target enrichment. Additionally, the quality and quantity of the recovered DNA vary depending on…

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Monday, October 21, 2019

Full-length isoform sequencing of the human MCF-7 cell line using PacBio long reads.

While advances in RNA sequencing methods have accelerated our understanding of the human transcriptome, isoform discovery remains a challenge because short read lengths require complicated assembly algorithms to infer the contiguity of full-length transcripts. With PacBio’s long reads, one can now sequence full-length transcript isoforms up to 10 kb. The PacBio Iso- Seq protocol produces reads that originate from independent observations of single molecules, meaning no assembly is needed. Here, we sequenced the transcriptome of the human MCF-7 breast cancer cell line using the Clontech SMARTer® cDNA preparation kit and the PacBio RS II. Using PacBio Iso-Seq bioinformatics software, we…

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Monday, October 21, 2019

Analysis of full-length metagenomic 16S genes by Single Molecule, Real-Time Sequencing

High-throughput sequencing of the complete 16S rRNA gene has become a valuable tool for characterizing microbial communities. However, the short reads produced by second-generation sequencing cannot provide taxonomic classification below the genus level. In this study, we demonstrate the capability of PacBio’s Single Molecule, Real-Time (SMRT) Sequencing to generate community profiles using mock microbial community samples from BEI Resources. We also evaluate multiplexing capabilities using PacBio barcodes on pooled samples comprising heterogeneous 16S amplicon populations representing soil, fecal, and mock communities.

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Monday, October 21, 2019

Building a platinum human genome assembly from single haplotype human genomes generated from long molecule sequencing

The human reference sequence has provided a foundation for studies of genome structure, human variation, evolutionary biology, and disease. At the time the reference was originally completed there were some loci recalcitrant to closure; however, the degree to which structural variation and diversity affected our ability to produce a representative genome sequence at these loci was still unknown. Many of these regions in the genome are associated with large, repetitive sequences and exhibit complex allelic diversity such producing a single, haploid representation is not possible. To overcome this challenge, we have sequenced DNA from two hydatidiform moles (CHM1 and CHM13),…

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Monday, October 21, 2019

Comprehensive genome and transcriptome structural analysis of a breast cancer cell line using PacBio long read sequencing

Genomic instability is one of the hallmarks of cancer, leading to widespread copy number variations, chromosomal fusions, and other structural variations. The breast cancer cell line SK-BR-3 is an important model for HER2+ breast cancers, which are among the most aggressive forms of the disease and affect one in five cases. Through short read sequencing, copy number arrays, and other technologies, the genome of SK-BR-3 is known to be highly rearranged with many copy number variations, including an approximately twenty-fold amplification of the HER2 oncogene. However, these technologies cannot precisely characterize the nature and context of the identified genomic events…

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Monday, October 21, 2019

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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Monday, October 21, 2019

Long-read assembly of the Aedes aegypti Aag2 cell line genome resolves ancient endogenous viral elements

Transmission of arboviruses such as Dengue Virus by Aedes aegypti causes debilitating disease across the globe. Disease in humans can include severe acute symptoms such as hemorrhagic fever and organ failure, but mosquitoes tolerate high titers of virus in a persistent infection. The mechanisms responsible for this viral tolerance are unclear. Recent publications highlighted the integration of genetic material from non-retroviral RNA viruses into the genome of the host during infection that relies upon endogenous retro-transcriptase activity from transposons. These endogenous viral elements (EVEs) found in the genome are predicted to be ancient, and at least some EVEs are under…

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Monday, October 21, 2019

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…

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Monday, October 21, 2019

Diploid genome assembly and comprehensive haplotype sequence reconstruction

Outside of the simplest cases (haploid, bacteria, or inbreds), genomic information is not carried in a single reference per individual, but rather has higher ploidy (n=>2) for almost all organisms. The existence of two or more highly related sequences within an individual makes it extremely difficult to build high quality, highly contiguous genome assemblies from short DNA fragments. Based on the earlier work on a polyploidy aware assembler, FALCON ( https://github.com/PacificBiosciences/FALCON) , we developed new algorithms and software (“FALCON-unzip”) for de novo haplotype reconstructions from SMRT Sequencing data. We generate two datasets for developing the algorithms and the prototype software:…

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Monday, October 21, 2019

Long-read assembly of the Aedes aegypti Aag2 cell line genome resolves ancient endogenous viral elements

Transmission of arboviruses such as Dengue and Zika viruses by Aedes aegypti causes widespread and debilitating disease across the globe. Disease in humans can include severe acute symptoms such as hemorrhagic fever, organ failure, and encephalitis; and yet, mosquitoes tolerate high titers of virus in a persistent infection. The mechanisms responsible for tolerance to viral infection in mosquitoes are still unclear. Recent publications have highlighted the integration of genetic material from non-retroviral RNA viruses into the genome of the host during infection that relies upon endogenous retro-transcriptase activity from transposons. These endogenous viral elements (EVEs) found in the genome are…

Read More »

Monday, October 21, 2019

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…

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Monday, October 21, 2019

Un-zipping diploid genomes – revealing all kinds of heterozygous variants from comprehensive hapltotig assemblies

Outside of the simplest cases (haploid, bacteria, or inbreds), genomic information is not carried in a single reference per individual, but rather has higher ploidy (n=>2) for almost all organisms. The existence of two or more highly related sequences within an individual makes it extremely difficult to build high quality, highly contiguous genome assemblies from short DNA fragments. Based on the earlier work on a polyploidy aware assembler, FALCON (https://github.com/PacificBiosciences/FALCON), we developed new algorithms and software (FALCON-unzip) for de novo haplotype reconstructions from SMRT Sequencing data. We apply the algorithms and the prototype software for (1) a highly repetitive diploid…

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Monday, October 21, 2019

Application specific barcoding strategies for SMRT Sequencing

Over the last few years, several advances were implemented in the PacBio RS II System to maximize throughput and efficiency while reducing the cost per sample. The number of useable bases per SMRT Cell now exceeds 1 Gb with the latest P6-C4 chemistry and 6-hour movies. For applications such as microbial sequencing, targeted sequencing, Iso-Seq (full-length isoform sequencing) and Nimblegen’s target enrichment method, current SMRT Cell yields could be an excess relative to project requirements. To this end, barcoding is a viable option for multiplexing samples. For microbial sequencing, multiplexing can be accomplished by tagging sheared genomic DNA during library…

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Monday, October 21, 2019

Multiplexing strategies for microbial whole genome SMRT Sequencing

The increased throughput of the RS II and Sequel Systems enables multiple microbes to be sequenced on a single SMRT Cell. This multiplexing can be readily achieved by simply incorporating a unique barcode for each microbe into the SMRTbell adapters after shearing genomic DNA using a streamlined library construction process. Incorporating a barcode without the requirement for PCR amplification prevents the loss of epigenetic information (e.g., methylation signatures), and the generation of chimeric sequences, while the modified protocol eliminates the need to build several individual SMRTbell libraries. We multiplexed up to 8 unique strains of H. pylori. Each strain was…

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