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Friday, February 26, 2021

Genome sequencing of microbial genomes using Single Molecule Real-time sequencing (SMRT) technology.

In the last year, high-throughput sequencing technologies have progressed from proof-of-concept to production quality. Although each technology is able to produce vast quantities of sequence information, in every case the underlying chemistry limits reads to very short lengths. We present a examining de novo assembly comparison with bacterial genome assembly varying genome size (from 3.1Mb to 7.6Mb) and different G+C contents (from 43% to 71%), respectively. We analyzed Solexa reads, 454 reads and Pacbio RS reads from Streptomyces sp. (Genome size, 7.6 Mb; G+C content, 71%), Psychrobacter sp. (Genome size, 3.5 Mb; G+C content, 43%), Salinibacterium sp. (Genome size, 3.1…

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Friday, February 26, 2021

Comparative genomics of Shiga toxin-producing Escherichia coli O145:H28 strains associated with the 2007 Belgium and 2010 US outbreaks.

Shiga toxin-producing Escherichia coli (STEC) is an emerging pathogen. Recently there has been a global in the number of outbreaks caused by non-O157 STECs, typically involving six serogroups O26, O45, 0103, 0111, and 0145. STEC O145:H28 has been associated with severe human disease including hemolytic-uremic syndrome (HUS), and is demonstrated by the 2007 Belgian ice-cream-associated outbreak and 2010 US lettuce-associated outbreak, with over 10% of patients developing HUS in each. The goal of this work was to do comparative genomics of strains, clinical and environmental, to investigate genome diversity and virulence evolution of this important foodborne pathogen.

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Friday, February 26, 2021

Genome sequencing of endosymbiotic bacterial Streptomyces sp. from Antartic lichen using Single Molecule Real-time Sequencing (SMRT) technology.

Along with the advent of next-generation sequencing (NGS) techniques, it has become possible to sequence a microbial genome very quickly with high coverage. Recently, PacificBioscience developed single molecule real-time sequencing (SMRT) technology, 3rd generation sequencing platform, which provide much longer (average read length: 1.5Kb) reads without PCR amplification. We did de novo sequencing of Streptomyces sp. using Illumina GAIIx, Roche 454 and PacBio RS system and compared the data. The endosymbiotic bacteria Streptomyces sp. PAMC 26508 was isolated from Antarctic lichen Psoroma sp. that grows attached rocks on Barton Peninsula, King George Island, Antarctica (62, 13’S, 58, 47’W). With 4…

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Friday, February 26, 2021

Advances in sequence consensus and clustering algorithms for effective de novo assembly and haplotyping applications.

One of the major applications of DNA sequencing technology is to bring together information that is distant in sequence space so that understanding genome structure and function becomes easier on a large scale. The Single Molecule Real Time (SMRT) Sequencing platform provides direct sequencing data that can span several thousand bases to tens of thousands of bases in a high-throughput fashion. In contrast to solving genomic puzzles by patching together smaller piece of information, long sequence reads can decrease potential computation complexity by reducing combinatorial factors significantly. We demonstrate algorithmic approaches to construct accurate consensus when the differences between reads…

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Friday, February 26, 2021

Automated, non-hybrid de novo genome assemblies and epigenomes of bacterial pathogens.

Understanding the genetic basis of infectious diseases is critical to enacting effective treatments, and several large-scale sequencing initiatives are underway to collect this information. Sequencing bacterial samples is typically performed by mapping sequence reads against genomes of known reference strains. While such resequencing informs on the spectrum of single-nucleotide differences relative to the chosen reference, it can miss numerous other forms of variation known to influence pathogenicity: structural variations (duplications, inversions), acquisition of mobile elements (phages, plasmids), homonucleotide length variation causing phase variation, and epigenetic marks (methylation, phosphorothioation) that influence gene expression to switch bacteria from non- pathogenic to pathogenic…

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Friday, February 26, 2021

Automated, non-hybrid de novo genome assemblies and epigenomes of bacterial pathogens

Understanding the genetic basis of infectious diseases is critical to enacting effective treatments, and several large-scale sequencing initiatives are underway to collect this information. Sequencing bacterial samples is typically performed by mapping sequence reads against genomes of known reference strains. While such resequencing informs on the spectrum of single nucleotide differences relative to the chosen reference, it can miss numerous other forms of variation known to influence pathogenicity: structural variations (duplications, inversions), acquisition of mobile elements (phages, plasmids), homonucleotide length variation causing phase variation, and epigenetic marks (methylation, phosphorothioation) that influence gene expression to switch bacteria from non-pathogenic to pathogenic…

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Friday, February 26, 2021

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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Friday, February 26, 2021

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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Friday, February 26, 2021

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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Friday, February 26, 2021

New discoveries from closing Salmonella genomes using Pacific Biosciences continuous long reads.

The newer hierarchical genome assembly process (HGAP) performs de novo assembly using data from a single PacBio long insert library. To assess the benefits of this method, DNA from several Salmonella enterica serovars was isolated from a pure culture. Genome sequencing was performed using Pacific Biosciences RS sequencing technology. The HGAP process enabled us to close sixteen Salmonella subsp. enterica genomes and their associated mobile elements: The ten serotypes include: Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) S. Bareilly, S. Heidelberg, S. Cubana, S. Javiana and S. Typhimurium, S. Newport, S. Montevideo, S. Agona, and S. Tennessee. In addition,…

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Friday, February 26, 2021

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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Friday, February 26, 2021

Near perfect de novo assemblies of eukaryotic genomes using PacBio long read sequencing.

Third generation single molecule sequencing technology from Pacific Biosciences, Moleculo, Oxford Nanopore, and other companies are revolutionizing genomics by enabling the sequencing of long, individual molecules of DNA and RNA. One major advantage of these technologies over current short read sequencing is the ability to sequence much longer molecules, thousands or tens of thousands of nucleotides instead of mere hundreds. This capacity gives researchers substantially greater power to probe into microbial, plant, and animal genomes, but it remains unknown on how to best use these data. To answer this, we systematically evaluated the human genome and 25 other important genomes…

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Friday, February 26, 2021

SMRT Sequencing solutions for investigative studies to understand evolutionary processes.

Single Molecule, Real-Time (SMRT) Sequencing holds promise for addressing new frontiers to understand molecular mechanisms in evolution and gain insight into adaptive strategies. With read lengths exceeding 10 kb, we are able to sequence high-quality, closed microbial genomes with associated plasmids, and investigate large genome complexities, such as long, highly repetitive, low-complexity regions and multiple tandem-duplication events. Improved genome quality, observed at 99.9999% (QV60) consensus accuracy, and significant reduction of gap regions in reference genomes (up to and beyond 50%) allow researchers to better understand coding sequences with high confidence, investigate potential regulatory mechanisms in noncoding regions, and make inferences…

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