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Tuesday, June 1, 2021

Complex alternative splicing patterns in hematopoietic cell subpopulations revealed by third-generation long reads.

Background: Alternative splicing expands the repertoire of gene functions and is a signature for different cell populations. Here we characterize the transcriptome of human bone marrow subpopulations including progenitor cells to understand their contribution to homeostasis and pathological conditions such as atherosclerosis and tumor metastasis. To obtain full-length transcript structures, we utilized long reads in addition to RNA-seq for estimating isoform diversity and abundance. Method: Freshly harvested, viable human bone marrow tissues were extracted from discarded harvesting equipment and separated into total bone marrow (total), lineage-negative (lin-) progenitor cells and differentiated cells (lin+) by magnetic bead sorting with antibodies to…

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Tuesday, June 1, 2021

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, June 1, 2021

Sequencing complex mixtures of HIV-1 genomes with single-base resolution.

A large number of distinct HIV-1 genomes can be present in a single clinical sample from a patient chronically infected with HIV-1. We examined samples containing complex mixtures of near-full-length HIV-1 genomes. Single molecules were sequenced as near-full-length (9.6 kb) amplicons directly from PCR products without shearing. Mathematical analysis techniques deconvolved the complex mixture of reads into estimates of distinct near-full-length viral genomes with their relative abundances. We correctly estimated the originating genomes to single-base resolution along with their relative abundances for mixtures where the truth was known exactly by independent sequencing methods. Correct estimates were made even when genomes…

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Tuesday, June 1, 2021

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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Tuesday, June 1, 2021

High-accuracy, single-base resolution of near-full-length HIV genomes.

Background: The HIV-1 proviral reservoir is incredibly stable, even while undergoing antiretroviral therapy, and is seen as the major barrier to HIV-1 eradication. Identifying and comprehensively characterizing this reservoir will be critical to achieving an HIV cure. Historically, this has been a tedious and labor intensive process, requiring high-replicate single-genome amplification reactions, or overlapping amplicons that are then reconstructed into full-length genomes by algorithmic imputation. Here, we present a deep sequencing and analysis method able to determine the exact identity and relative abundances of near-full-length HIV genomes from samples containing mixtures of genomes without shearing or complex bioinformatic reconstruction. Methods:…

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Tuesday, June 1, 2021

Metagenomes of native and electrode-enriched microbial communities from the Soudan Iron Mine.

Despite apparent carbon limitation, anoxic deep subsurface brines at the Soudan Underground Iron Mine harbor active microbial communities. To characterize these assemblages, we performed shotgun metagenomics of native and enriched samples. Following enrichment on poised electrodes and long read sequencing, we recovered from the metagenome the closed, circular genome of a novel Desulfuromonas sp. with remarkable genomic features that were not fully resolved by short read assembly alone. This organism was essentially absent in unenriched Soudan communities, indicating that electrodes are highly selective for putative metal reducers. Native community metagenomes suggest that carbon cycling is driven by methyl-C1 metabolism, in…

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Tuesday, June 1, 2021

Barcoding strategies for multiplexing of samples using a long-read sequencing technology.

We have developed barcoding reagents and workflows for multiplexing amplicons or fragmented native genomic (DNA) prior to Single Molecule, Real-Time (SMRT) Sequencing. The long reads of PacBio’s SMRT Sequencing enable detection of linked mutations across multiple kilobases (kb) of sequence. This feature is particularly useful in the context of mutational analysis or SNP confirmation, where a large number of samples are generated routinely. To validate this workflow, a set of 384 1.7-kb amplicons, each derived from variants of the Phi29 DNA polymerase gene, were barcoded during amplification, pooled, and sequenced on a single SMRT Cell. To demonstrate the applicability of…

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Tuesday, June 1, 2021

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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Tuesday, June 1, 2021

Genome and transcriptome of the refeneration-competent flatworm, Macrostomum lignano

The free-living flatworm, Macrostomum lignano, much like its better known planarian relative, Schmidtea mediterranea, has an impressive regenerative capacity. Following injury, this species has the ability to regenerate almost an entirely new organism. This is attributable to the presence of an abundant somatic stem cell population, the neoblasts. These cells are also essential for the ongoing maintenance of most tissues, as their loss leads to irreversible degeneration of the animal. This set of unique properties makes a subset of flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cell fate specification, and regeneration. The use of…

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Tuesday, June 1, 2021

Improving the goat long-read assembly with optical mapping and Hi-C scaffolding

Reference genome assemblies provide important context in genetics by standardizing the order of genes and providing a universal set of coordinates for individual nucleotides. Often due to the high complexity of genic regions and higher copy number of genes involved in immune function, immunity-related genes are often misassembled in current reference assemblies. This problem is particularly ubiquitous in the reference genomes of non-model organisms as they often do not receive the years of curation necessary to resolve annotation and assembly errors. In this study, we reassemble a reference genome of the goat (Capra hircus) using modern PacBio technology in tandem…

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Tuesday, June 1, 2021

A comprehensive study of the sugar pine (Pinus lambertiana) transcriptome implemented through diverse next-generation sequencing approaches

The assembly, annotation, and characterization of the sugar pine (Pinus lambertiana Dougl.) transcriptome represents an opportunity to study the genetic mechanisms underlying resistance to the invasive white pine blister rust (Cronartium ribicola) as well as responses to other abiotic stresses. The assembled transcripts also provide a resource to improve the genome assembly. We selected a diverse set of tissues allowing the first comprehensive evaluation of the sugar pine gene space. We have combined short read sequencing technologies (Illumina MiSeq and HiSeq) with the relatively new Pacific Biosciences Iso-Seq approach. From the 2.5 billion and 1.6 million Illumina and PacBio (46…

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Tuesday, June 1, 2021

A comprehensive lincRNA analysis: From conifers to trees

We have produced an updated annotation of the Norway spruce genome on the basis of an in siliconormalised set of RNA-Seq data obtained from 1,529 samples and comprising 15.5 billion paired-end Illumina HiSeq reads complemented by 18Mbp of PacBio cDNA data (3.2M sequences). In addition to augmenting and refining the previous protein coding gene annotation, here we focus on the addition of long intergenic non-coding RNA (lincRNA) and micro RNA (miRNA) genes. In addition to non-coding loci, our analyses also identified protein coding genes that had been missed by the initial genome annotation and enabled us to update the annotation…

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Tuesday, June 1, 2021

How to Compare and Cluster Every Known Genome in about an Hour

Given a massive collection of sequences, it is infeasible to perform pairwise alignment for basic tasks like sequence clustering and search. To address this problem, we demonstrate that the MinHash technique, first applied to clustering web pages, can be applied to biological sequences with similar effect, and extend this idea to include biologically relevant distance and significance measures. Our new tool, Mash, uses MinHash locality-sensitive hashing to reduce large sequences to a representative sketch and rapidly estimate pairwise distances between genomes or metagenomes. Using Mash, we explored several use cases, including a 5,000-fold size reduction and clustering of all 55,000…

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