Analysis of gene expression for microminipig liver transcriptomes using parallel long-read technology and short-read sequencing

AbstractThe growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as potential sources of new antimicrobials. PCR amplicon analyses and short-read sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes, indicating their potential for producing novel and useful compounds. However, recovering full-length BGC sequences from uncultivated bacteria remains a challenge due to the technological restraints of short-read sequencing, thus making assessment of BGC diversity difficult. Here, long-read sequencing and genome mining were used to recover >1400 mostly full-length BGCs that demonstrate the rich diversity of BGCs from uncultivated lineages present in soil from Mars Oasis, Antarctica. A large number of highly divergent BGCs were not only found in the phyla Acidobacteriota, Verrucomicrobiota and Gemmatimonadota but also in the actinobacterial classes Acidimicrobiia and Thermoleophilia and the gammaproteobacterial order UBA7966. The latter furthermore contained a potential novel family of RiPPs. Our findings underline the biosynthetic potential of underexplored phyla as well as unexplored lineages within seemingly well-studied producer phyla. They also showcase long-read metagenomic sequencing as a promising way to access the untapped genetic reservoir of specialised metabolite gene clusters of the uncultured majority of microbes.

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Rapid Mycobacterium tuberculosis spoligotyping from uncorrected long reads using Galru

10.1101/2020.05.31.126490 ◽

2020 ◽

Author(s):

Andrew J. Page ◽

Nabil-Fareed Alikhan ◽

Michael Strinden ◽

Thanh Le Viet ◽

Timofey Skvortsov

Keyword(s):

Mycobacterium Tuberculosis ◽

State Of The Art ◽

Sequence Data ◽

Human Pathogen ◽

Sequencing Data ◽

Short Read ◽

Short Read Sequencing ◽

Long Reads ◽

Long Read

AbstractSpoligotyping of Mycobacterium tuberculosis provides a subspecies classification of this major human pathogen. Spoligotypes can be predicted from short read genome sequencing data; however, no methods exist for long read sequence data such as from Nanopore or PacBio. We present a novel software package Galru, which can rapidly detect the spoligotype of a Mycobacterium tuberculosis sample from as little as a single uncorrected long read. It allows for near real-time spoligotyping from long read data as it is being sequenced, giving rapid sample typing. We compare it to the existing state of the art software and find it performs identically to the results obtained from short read sequencing data. Galru is freely available from https://github.com/quadram-institute-bioscience/galru under the GPLv3 open source licence.

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Closed Genome Sequence of Salmonella enterica Serovar Richmond Strain CFSAN000191, Obtained with Nanopore Sequencing

Microbiology Resource Announcements ◽

10.1128/mra.01472-18 ◽

2018 ◽

Vol 7 (23) ◽

Cited By ~ 3

Author(s):

Narjol González-Escalona ◽

Kuan Yao ◽

Maria Hoffmann

Keyword(s):

Genome Sequence ◽

Salmonella Enterica ◽

Nanopore Sequencing ◽

Short Read ◽

Content Type ◽

Short Read Sequencing ◽

Long Read

Here we report the genome sequence of Salmonella enterica serovar Richmond strain CFSAN000191, isolated from tilapia from Thailand in 2005. The genome was determined by a combination of long-read and short-read sequencing.

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Closed Genome Sequences of Three Salmonella enterica Strains Belonging to Serovars Saintpaul, Weltevreden, and Thompson, Isolated from Mexico

Microbiology Resource Announcements ◽

10.1128/mra.00656-19 ◽

2019 ◽

Vol 8 (28) ◽

Cited By ~ 2

Author(s):

Narjol Gonzalez-Escalona ◽

J. R. Aguirre-Sánchez ◽

J. R. Ibarra-Rodríguez ◽

C. Chaidez-Quiroz ◽

Jaime Martinez-Urtaza

Keyword(s):

River Water ◽

Salmonella Enterica ◽

Genome Sequences ◽

Short Read ◽

Content Type ◽

Short Read Sequencing ◽

Long Read ◽

Sequence Types

Here, we report the genome sequences of three Salmonella enterica strains belonging to serovars Weltevreden (CFSAN047349), Saintpaul (CFSAN047351), and Thompson (CFSAN047352), isolated from river water in Sinaloa, Mexico. The genomes were closed by a combination of long-read and short-read sequencing. The strain sequence types (STs) are ST365, ST50, and ST26, respectively.

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Complete Genome Sequence of Rubrobacter xylanophilus Strain AA3-22, Isolated from Arima Onsen in Japan

Microbiology Resource Announcements ◽

10.1128/mra.00818-19 ◽

2019 ◽

Vol 8 (34) ◽

Cited By ~ 1

Author(s):

Natsuki Tomariguchi ◽

Kentaro Miyazaki

Keyword(s):

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Hot Spring ◽

Sequencing Data ◽

Short Read ◽

Content Type ◽

Short Read Sequencing ◽

Oxford Nanopore ◽

Long Read

Rubrobacter xylanophilus strain AA3-22, belonging to the phylum Actinobacteria, was isolated from nonvolcanic Arima Onsen (hot spring) in Japan. Here, we report the complete genome sequence of this organism, which was obtained by combining Oxford Nanopore long-read and Illumina short-read sequencing data.

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Measuring differential gene expression by short read sequencing: quantitative comparison to 2-channel gene expression microarrays

BMC Genomics ◽

10.1186/1471-2164-10-221 ◽

2009 ◽

Vol 10 (1) ◽

pp. 221 ◽

Cited By ~ 111

Author(s):

Joshua S Bloom ◽

Zia Khan ◽

Leonid Kruglyak ◽

Mona Singh ◽

Amy A Caudy

Keyword(s):

Gene Expression ◽

Differential Gene Expression ◽

Quantitative Comparison ◽

Short Read ◽

Short Read Sequencing ◽

Gene Expression Microarrays ◽

Channel Gene ◽

Differential Gene ◽

Expression Microarrays

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Towards a Comprehensive Variation Benchmark for Challenging Medically-Relevant Autosomal Genes

10.1101/2021.06.07.444885 ◽

2021 ◽

Author(s):

Justin Wagner ◽

Nathan D Olson ◽

Lindsay Harris ◽

Jennifer McDaniel ◽

Haoyu Cheng ◽

...

Keyword(s):

Genome Sequencing ◽

Method Development ◽

Clinical Applications ◽

Accurate Analysis ◽

Short Read ◽

Short Read Sequencing ◽

Repetitive Nature ◽

Long Read ◽

Comprehensive Characterization

The repetitive nature and complexity of multiple medically important genes make them intractable to accurate analysis, despite the maturity of short-read sequencing, resulting in a gap in clinical applications of genome sequencing. The Genome in a Bottle Consortium has provided benchmark variant sets, but these excluded some medically relevant genes due to their repetitiveness or polymorphic complexity. In this study, we characterize 273 of these 395 challenging autosomal genes that have multiple implications for medical sequencing. This extended, curated benchmark reports over 17,000 SNVs, 3,600 INDELs, and 200 SVs each for GRCh37 and GRCh38. We show that false duplications in either GRCh37 or GRCh38 result in reference-specific, missed variants for short- and long-read technologies in medically important genes including CBS, CRYAA, and KCNE1. Our proposed solution improves variant recall in these genes from 8% to 100%. This benchmark will significantly improve the comprehensive characterization of these medically relevant genes and guide new method development.

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LinkedSV for detection of mosaic structural variants from linked-read exome and genome sequencing data

10.1101/409789 ◽

2018 ◽

Cited By ~ 2

Author(s):

Li Fang ◽

Charlly Kao ◽

Michael V Gonzalez ◽

Fernanda A Mafra ◽

Renata Pellegrino da Silva ◽

...

Keyword(s):

Exome Sequencing ◽

Read Depth ◽

Structural Variants ◽

Sequencing Data ◽

High Coverage ◽

Short Read ◽

Short Read Sequencing ◽

Sequencing Studies ◽

Long Read ◽

Local Assembly

AbstractLinked-read sequencing provides long-range information on short-read sequencing data by barcoding reads originating from the same DNA molecule, and can improve the detection and breakpoint identification for structural variants (SVs). We present LinkedSV for SV detection on linked-read sequencing data. LinkedSV considers barcode overlapping and enriched fragment endpoints as signals to detect large SVs, while it leverages read depth, paired-end signals and local assembly to detect small SVs. Benchmarking studies demonstrates that LinkedSV outperforms existing tools, especially on exome data and on somatic SVs with low variant allele frequencies. We demonstrate clinical cases where LinkedSV identifies disease causal SVs from linked-read exome sequencing data missed by conventional exome sequencing, and show examples where LinkedSV identifies SVs missed by high-coverage long-read sequencing. In summary, LinkedSV can detect SVs missed by conventional short-read and long-read sequencing approaches, and may resolve negative cases from clinical genome/exome sequencing studies.

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Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease

10.1101/176651 ◽

2018 ◽

Cited By ~ 1

Author(s):

Mark T. W. Ebbert ◽

Stefan Farrugia ◽

Jonathon Sens ◽

Karen Jansen-West ◽

Tania F. Gendron ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Repeat Expansion ◽

Whole Genome ◽

Short Read ◽

Short Read Sequencing ◽

Sequencing Technologies ◽

Long Read ◽

Repeat Expansions ◽

Targeted Approach

AbstractBackground: Many neurodegenerative diseases are caused by nucleotide repeat expansions, but most expansions, like the C9orf72 ‘GGGGCC’ (G4C2) repeat that causes approximately 5-7% of all amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases, are too long to sequence using short-read sequencing technologies. It is unclear whether long-read sequencing technologies can traverse these long, challenging repeat expansions. Here, we demonstrate that two long-read sequencing technologies, Pacific Biosciences’ (PacBio) and Oxford Nanopore Technologies’ (ONT), can sequence through disease-causing repeats cloned into plasmids, including the FTD/ALS-causing G4C2 repeat expansion. We also report the first long-read sequencing data characterizing the C9orf72 G4C2 repeat expansion at the nucleotide level in two symptomatic expansion carriers using PacBio whole-genome sequencing and a no-amplification (No-Amp) targeted approach based on CRISPR/Cas9.Results: Both the PacBio and ONT platforms successfully sequenced through the repeat expansions in plasmids. Throughput on the MinlON was a challenge for whole-genome sequencing; we were unable to attain reads covering the human C9orf72 repeat expansion using 15 flow cells. We obtained 8x coverage across the C9orf72 locus using the PacBio Sequel, accurately reporting the unexpanded allele at eight repeats, and reading through the entire expansion with 1324 repeats (7941 nucleotides). Using the No-Amp targeted approach, we attained >800x coverage and were able to identify the unexpanded allele, closely estimate expansion size, and assess nucleotide content in a single experiment. We estimate the individual’s repeat region was >99% G4C2 content, though we cannot rule out small interruptions.Conclusions: Our findings indicate that long-read sequencing is well suited to characterizing known repeat expansions, and for discovering new disease-causing, disease-modifying, or risk-modifying repeat expansions that have gone undetected with conventional short-read sequencing. The PacBio No-Amp targeted approach may have future potential in clinical and genetic counseling environments. Larger and deeper long-read sequencing studies in C9orf72 expansion carriers will be important to determine heterogeneity and whether the repeats are interrupted by non-G4C2 content, potentially mitigating or modifying disease course or age of onset, as interruptions are known to do in other repeat-expansion disorders. These results have broad implications across all diseases where the genetic etiology remains unclear.

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