Assessing the utility of long-read nanopore sequencing for rapid and efficient characterization of mobile element insertions

ABSTRACTWater bodies around the world are increasingly threatened by harmful algal blooms (HABs) under current trends of rising water temperature and nutrient load. Metagenomic characterization of HABs can be combined with water quality and environmental data to better understand and predict the occurrence of toxic events. However, standard short-read sequencing typically yields highly fragmented metagenomes, preventing direct connection of genes to a single genome. Using Oxford Nanopore long-read sequencing, we were able to obtain high quality metagenome-assembled genomes, and show that dominant organisms in a HAB are readily identified, though different analyses disagreed on the identity of rare taxa. Genes from diverse functional categories were found not only in the most dominant genera, but also in several less common ones. Using simulated datasets, we show that the Flongle flowcell may provide an option for HAB monitoring with less data, at the expense of failing to detect rarer organisms and increasing fragmentation of the metagenome. Based on these results, we believe that Nanopore sequencing provides a fast, portable, and affordable method for studying HABs.

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Long-read Time-course Profiling of the Host Cell Response to Herpesvirus Infection Using Nanopore and Synthetic Long-Read Transcriptome Sequencing

10.21203/rs.3.rs-264666/v1 ◽

2021 ◽

Author(s):

Zoltán Maróti ◽

Norbert Moldován ◽

Gábor Torma ◽

Victoria A. Jefferson ◽

Zsolt Csabai ◽

...

Keyword(s):

Gene Networks ◽

Time Course ◽

Average Distance ◽

Host Cells ◽

Nanopore Sequencing ◽

Splice Isoforms ◽

Transcript Isoforms ◽

First Report ◽

Long Read

Abstract Third-generation sequencing is able to read full-length transcripts and thus to efficiently identify RNA molecules and transcript isoforms, including transcript length and splice isoforms. In this study, we report the time-course profiling of the effect of bovine alphaherpesvirus type 1 on the gene expression of bovine epithelial cells using direct cDNA sequencing carried out on MinION device of Oxford Nanopore Technologies. These investigations revealed a substantial up- and down-regulatory effect of the virus on several gene networks of the host cells, including those that are associated with antiviral response, as well as with viral transcription and translation. Additionally, we report a large number of novel bovine transcripts identified by nanopore and synthetic long-read sequencing. This study demonstrates that viral infection does not lead to a change in the average distance between promoters and transcription start sites, and between polyadenylation signals and transcription end sites. However, it causes differential expression of transcript isoforms. We could not detect an increased rate of transcriptional readthroughs as described in another alphaherpesvirus. According to our knowledge, this is the first report on the use of LoopSeq for the analysis of eukaryotic transcriptomes. This is also the first report on the application of nanopore sequencing for the kinetic characterization of cellular transcriptomes. This study also demonstrates the utility of nanopore sequencing for the characterization of dynamic changes of transcriptomes in any organisms.

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Precise characterization of somatic structural variations and mobile element insertions from paired long-read sequencing data with nanomonsv

10.1101/2020.07.22.214262 ◽

2020 ◽

Author(s):

Yuichi Shiraishi ◽

Junji Koya ◽

Kenichi Chiba ◽

Yuki Saito ◽

Ai Okada ◽

...

Keyword(s):

Matched Control ◽

Mobile Element ◽

Sequencing Data ◽

Structural Variations ◽

Short Read ◽

Long Read ◽

Functional Consequences ◽

Single Base Resolution ◽

Mutational Processes

AbstractWe introduce our novel software, nanomonsv, for detecting somatic structural variations (SVs) using tumor and matched control long-read sequencing data with a single-base resolution. Using paired long-read sequencing data from three cancer cell-lines and their matched lymphoblastoid lines, we demonstrate that our approach can identify not only somatic SVs that can be captured with short-read technologies but also novel ones especially those whose breakpoints are located in repeat regions. In addition, we have developed a workflow for classifying mobile element insertions while elucidating their in-depth properties such as 5′ truncations, internal inversion as well as source sites in the case of LINE1 transductions. Finally, we identify complex SVs probably caused by replication mechanisms or telomere crisis by examining the co-occurrence of multiple somatic SVs in common supporting reads. In summary, our approaches applied to cancer long-read sequencing data can reveal various features of somatic SVs and will lead to further understanding of mutational processes and functional consequences of somatic SVs.

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Cas9 targeted enrichment of mobile elements using nanopore sequencing

10.1101/2021.02.10.430605 ◽

2021 ◽

Author(s):

Torrin L. McDonald ◽

Weichen Zhou ◽

Christopher Castro ◽

Camille Mumm ◽

Jessica A. Switzenberg ◽

...

Keyword(s):

Genetic Disorders ◽

Mobile Element ◽

Flow Cell ◽

Read Length ◽

Nanopore Sequencing ◽

Short Read Sequencing ◽

Human Genomes ◽

Long Read ◽

Genomic Regions ◽

Targeted Enrichment

AbstractMobile element insertions (MEIs) are highly repetitive genomic sequences that contribute to inter- and intra-individual genetic variation and can lead to genetic disorders. Targeted and whole-genome approaches using short-read sequencing have been developed to identify reference and non-reference MEIs; however, the read length hampers detection of these elements in complex genomic regions. Here, we pair Cas9 targeted nanopore sequencing with computational methodologies to capture active MEIs in human genomes. We demonstrate parallel enrichment for distinct classes of MEIs, averaging 44% of reads on targeted signals. We show an individual flow cell can recover a remarkable fraction of MEIs (97% L1Hs, 93% AluYb, 51% AluYa, 99% SVA_F, and 65% SVA_E). We identify twenty-one non-reference MEIs in GM12878 overlooked by modern, long-read analysis pipelines, primarily in repetitive genomic regions. This work introduces the utility of nanopore sequencing for MEI enrichment and lays the foundation for rapid discovery of elusive, repetitive genetic elements.

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Cas9 targeted enrichment of mobile elements using nanopore sequencing

Nature Communications ◽

10.1038/s41467-021-23918-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Torrin L. McDonald ◽

Weichen Zhou ◽

Christopher P. Castro ◽

Camille Mumm ◽

Jessica A. Switzenberg ◽

...

Keyword(s):

Genetic Disorders ◽

Mobile Element ◽

Read Length ◽

Whole Genome ◽

Nanopore Sequencing ◽

Short Read Sequencing ◽

Human Genomes ◽

Long Read ◽

Genomic Regions ◽

Targeted Enrichment

AbstractMobile element insertions (MEIs) are repetitive genomic sequences that contribute to genetic variation and can lead to genetic disorders. Targeted and whole-genome approaches using short-read sequencing have been developed to identify reference and non-reference MEIs; however, the read length hampers detection of these elements in complex genomic regions. Here, we pair Cas9-targeted nanopore sequencing with computational methodologies to capture active MEIs in human genomes. We demonstrate parallel enrichment for distinct classes of MEIs, averaging 44% of reads on-targeted signals and exhibiting a 13.4-54x enrichment over whole-genome approaches. We show an individual flow cell can recover most MEIs (97% L1Hs, 93% AluYb, 51% AluYa, 99% SVA_F, and 65% SVA_E). We identify seventeen non-reference MEIs in GM12878 overlooked by modern, long-read analysis pipelines, primarily in repetitive genomic regions. This work introduces the utility of nanopore sequencing for MEI enrichment and lays the foundation for rapid discovery of elusive, repetitive genetic elements.

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Time course profiling of host cell response to herpesvirus infection using nanopore and synthetic long-read transcriptome sequencing

Scientific Reports ◽

10.1038/s41598-021-93142-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Zoltán Maróti ◽

Dóra Tombácz ◽

Norbert Moldován ◽

Gábor Torma ◽

Victoria A. Jefferson ◽

...

Keyword(s):

Gene Networks ◽

Time Course ◽

Host Cells ◽

Nanopore Sequencing ◽

Splice Isoforms ◽

Transcript Isoforms ◽

First Report ◽

Host Cell Response ◽

Long Read

AbstractThird-generation sequencing is able to read full-length transcripts and thus to efficiently identify RNA molecules and transcript isoforms, including transcript length and splice isoforms. In this study, we report the time-course profiling of the effect of bovine alphaherpesvirus type 1 on the gene expression of bovine epithelial cells using direct cDNA sequencing carried out on MinION device of Oxford Nanopore Technologies. These investigations revealed a substantial up- and down-regulatory effect of the virus on several gene networks of the host cells, including those that are associated with antiviral response, as well as with viral transcription and translation. Additionally, we report a large number of novel bovine transcript isoforms identified by nanopore and synthetic long-read sequencing. This study demonstrates that viral infection causes differential expression of host transcript isoforms. We could not detect an increased rate of transcriptional readthroughs as described in another alphaherpesvirus. According to our knowledge, this is the first report on the use of LoopSeq for the analysis of eukaryotic transcriptomes. This is also the first report on the application of nanopore sequencing for the kinetic characterization of cellular transcriptomes. This study also demonstrates the utility of nanopore sequencing for the characterization of dynamic transcriptomes in any organisms.

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kangaroo, a Mobile Element From Volvox carteri, Is a Member of a Newly Recognized Third Class of Retrotransposons

Genetics ◽

10.1093/genetics/162.4.1617 ◽

2002 ◽

Vol 162 (4) ◽

pp. 1617-1630

Author(s):

Leonard Duncan ◽

Kristine Bouckaert ◽

Fay Yeh ◽

David L Kirk

Keyword(s):

Genomic Structure ◽

Mobile Element ◽

Direct Repeat ◽

Structural Features ◽

Volvox Carteri ◽

Developmentally Regulated ◽

Closed Circle ◽

Integration Mechanisms ◽

And Function

Abstract Retrotransposons play an important role in the evolution of genomic structure and function. Here we report on the characterization of a novel retrotransposon called kangaroo from the multicellular green alga, Volvox carteri. kangaroo elements are highly mobile and their expression is developmentally regulated. They probably integrate via double-stranded, closed-circle DNA intermediates through the action of an encoded recombinase related to the λ-site-specific integrase. Phylogenetic analysis indicates that kangaroo elements are closely related to other unorthodox retrotransposons including PAT (from a nematode), DIRS-1 (from Dictyostelium), and DrDIRS1 (from zebrafish). PAT and kangaroo both contain split direct repeat (SDR) termini, and here we show that DIRS-1 and DrDIRS1 elements contain terminal features structurally related to SDRs. Thus, these mobile elements appear to define a third class of retrotransposons (the DIRS1 group) that are unified by common structural features, genes, and integration mechanisms, all of which differ from those of LTR and conventional non-LTR retrotransposons.

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MBRS-47. RAPID MOLECULAR SUBGROUPING OF MEDULLOBLASTOMA BASED ON DNA METHYLATION BY NANOPORE SEQUENCING

Neuro-Oncology ◽

10.1093/neuonc/noaa222.556 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii406-iii406

Author(s):

Julien Masliah-Planchon ◽

Elodie Girard ◽

Philipp Euskirchen ◽

Christine Bourneix ◽

Delphine Lequin ◽

...

Keyword(s):

Dna Methylation ◽

Single Molecule ◽

Nanopore Sequencing ◽

Molecular Subgroup ◽

Group Assignment ◽

Group 4 ◽

Methylation Assay ◽

Tumor Group ◽

Long Read ◽

Group 3

Abstract Medulloblastoma (MB) can be classified into four molecular subgroups (WNT group, SHH group, group 3, and group 4). The gold standard of assignment of molecular subgroup through DNA methylation profiling uses Illumina EPIC array. However, this tool has some limitation in terms of cost and timing, in order to get the results soon enough for clinical use. We present an alternative DNA methylation assay based on nanopore sequencing efficient for rapid, cheaper, and reliable subgrouping of clinical MB samples. Low-depth whole genome with long-read single-molecule nanopore sequencing was used to simultaneously assess copy number profile and MB subgrouping based on DNA methylation. The DNA methylation data generated by Nanopore sequencing were compared to a publicly available reference cohort comprising over 2,800 brain tumors including the four subgroups of MB (Capper et al. Nature; 2018) to generate a score that estimates a confidence with a tumor group assignment. Among the 24 MB analyzed with nanopore sequencing (six WNT, nine SHH, five group 3, and four group 4), all of them were classified in the appropriate subgroup established by expression-based Nanostring subgrouping. In addition to the subgrouping, we also examine the genomic profile. Furthermore, all previously identified clinically relevant genomic rearrangements (mostly MYC and MYCN amplifications) were also detected with our assay. In conclusion, we are confirming the full reliability of nanopore sequencing as a novel rapid and cheap assay for methylation-based MB subgrouping. We now plan to implement this technology to other embryonal tumors of the central nervous system.

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Nanopore sequencing of single-cell transcriptomes with scCOLOR-seq

Nature Biotechnology ◽

10.1038/s41587-021-00965-w ◽

2021 ◽

Author(s):

Martin Philpott ◽

Jonathan Watson ◽

Anjan Thakurta ◽

Tom Brown ◽

...

Keyword(s):

Single Cell ◽

Error Detection ◽

Single Cells ◽

Fusion Transcript ◽

Building Blocks ◽

Myeloma Cell ◽

Nanopore Sequencing ◽

Long Read ◽

Unique Molecular Identifier ◽

Transcript Detection

AbstractHere we describe single-cell corrected long-read sequencing (scCOLOR-seq), which enables error correction of barcode and unique molecular identifier oligonucleotide sequences and permits standalone cDNA nanopore sequencing of single cells. Barcodes and unique molecular identifiers are synthesized using dimeric nucleotide building blocks that allow error detection. We illustrate the use of the method for evaluating barcode assignment accuracy, differential isoform usage in myeloma cell lines, and fusion transcript detection in a sarcoma cell line.

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Nanopore-Sequencing Characterization of the Gut Microbiota of Melolontha melolontha Larvae: Contribution to Protection against Entomopathogenic Nematodes?

Pathogens ◽

10.3390/pathogens10040396 ◽

2021 ◽

Vol 10 (4) ◽

pp. 396

Author(s):

Ewa Sajnaga ◽

Marcin Skowronek ◽

Agnieszka Kalwasińska ◽

Waldemar Kazimierczak ◽

Karolina Ferenc ◽

...

Keyword(s):

Gut Microbiota ◽

Entomopathogenic Nematodes ◽

Bacterial Species ◽

Antagonistic Activity ◽

Rrna Gene ◽

Nanopore Sequencing ◽

Bacterial Phyla ◽

Melolontha Melolontha

This study focused on the potential relationships between midgut microbiota of the common cockchafer Melolontha melolontha larvae and their resistance to entomopathogenic nematodes (EPN) infection. We investigated the bacterial community associated with control and unsusceptible EPN-exposed insects through nanopore sequencing of the 16S rRNA gene. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the most abundant bacterial phyla within the complex and variable midgut microbiota of the wild M. melolontha larvae. The core microbiota was found to include 82 genera, which accounted for 3.4% of the total number of identified genera. The EPN-resistant larvae differed significantly from the control ones in the abundance of many genera belonging to the Actinomycetales, Rhizobiales, and Clostridiales orders. Additionally, the analysis of the microbiome networks revealed different sets of keystone midgut bacterial genera between these two groups of insects, indicating differences in the mutual interactions between bacteria. Finally, we detected Xenorhabdus and Photorhabdus as gut residents and various bacterial species exhibiting antagonistic activity against these entomopathogens. This study paves the way to further research aimed at unravelling the role of the host gut microbiota on the output of EPN infection, which may contribute to enhancement of the efficiency of nematodes used in eco-friendly pest management.

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