Single-molecule long-read sequencing reveals a conserved intact long RNA profile in sperm

AbstractSperm contributes diverse RNAs to the zygote. While sperm small RNAs have been shown to impact offspring phenotypes, our knowledge of the sperm transcriptome, especially the composition of long RNAs, has been limited by the lack of sensitive, high-throughput experimental techniques that can distinguish intact RNAs from fragmented RNAs, known to abound in sperm. Here, we integrate single-molecule long-read sequencing with short-read sequencing to detect sperm intact RNAs (spiRNAs). We identify 3440 spiRNA species in mice and 4100 in humans. The spiRNA profile consists of both mRNAs and long non-coding RNAs, is evolutionarily conserved between mice and humans, and displays an enrichment in mRNAs encoding for ribosome. In sum, we characterize the landscape of intact long RNAs in sperm, paving the way for future studies on their biogenesis and functions. Our experimental and bioinformatics approaches can be applied to other tissues and organisms to detect intact transcripts.

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Transcriptome innovations in primates revealed by single-molecule long-read sequencing

10.1101/2021.11.10.468034 ◽

2021 ◽

Author(s):

Luis Ferrandez-Peral ◽

Xiaoyu Zhan ◽

Marina Alvarez-Estape ◽

Cristina Chiva ◽

Paula Esteller-Cucala ◽

...

Keyword(s):

Single Molecule ◽

Great Apes ◽

Primate Evolution ◽

Lymphoblastoid Cell Lines ◽

Short Read ◽

Limited Impact ◽

Short Read Sequencing ◽

Important Target ◽

Long Read ◽

Novel Transcripts

Transcriptomic diversity greatly contributes to the fundamentals of disease, lineage-specific biology, and environmental adaptation. However, much of the actual isoform repertoire contributing to shaping primate evolution remains unknown. Here, we combined deep long- and short-read sequencing complemented with mass spectrometry proteomics in a panel of lymphoblastoid cell lines (LCLs) from human, three other great apes, and rhesus macaque, producing the largest full-length isoform catalog in primates to date. Our transcriptomes reveal thousands of novel transcripts, some of them under active translation, expanding and completing the repertoire of primate gene models. Our comparative analyses unveil hundreds of transcriptomic innovations and isoform usage changes related to immune function and immunological disorders. The confluence of these innovations with signals of positive selection and their limited impact in the proteome points to changes in alternative splicing in genes involved in immune response as an important target of recent regulatory divergence in primates.

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Perspectives and benefits of high-throughput long-read sequencing in microbial ecology

Applied and Environmental Microbiology ◽

10.1128/aem.00626-21 ◽

2021 ◽

Author(s):

Leho Tedersoo ◽

Mads Albertsen ◽

Sten Anslan ◽

Benjamin Callahan

Keyword(s):

Microbial Ecology ◽

High Throughput ◽

Single Molecule ◽

High Throughput Sequencing ◽

Environmental Dna ◽

Nanopore Sequencing ◽

High Quality ◽

Short Read ◽

Sequencing Technologies ◽

Long Read

Short-read, high-throughput sequencing (HTS) methods have yielded numerous important insights into microbial ecology and function. Yet, in many instances short-read HTS techniques are suboptimal, for example by providing insufficient phylogenetic resolution or low integrity of assembled genomes. Single-molecule and synthetic long-read (SLR) HTS methods have successfully ameliorated these limitations. In addition, nanopore sequencing has generated a number of unique analysis opportunities such as rapid molecular diagnostics and direct RNA sequencing, and both PacBio and nanopore sequencing support detection of epigenetic modifications. Although initially suffering from relatively low sequence quality, recent advances have greatly improved the accuracy of long read sequencing technologies. In spite of great technological progress in recent years, the long-read HTS methods (PacBio and nanopore sequencing) are still relatively costly, require large amounts of high-quality starting material, and commonly need specific solutions in various analysis steps. Despite these challenges, long-read sequencing technologies offer high-quality, cutting-edge alternatives for testing hypotheses about microbiome structure and functioning as well as assembly of eukaryote genomes from complex environmental DNA samples.

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Phased Haplotype Resolution of the SLC6A4 Promoter Using Long-Read Single Molecule Real-Time (SMRT) Sequencing

Genes ◽

10.3390/genes11111333 ◽

2020 ◽

Vol 11 (11) ◽

pp. 1333

Author(s):

Mariana R. Botton ◽

Yao Yang ◽

Erick R. Scott ◽

Robert J. Desnick ◽

Stuart A. Scott

Keyword(s):

Real Time ◽

Single Molecule ◽

Variable Number ◽

Sequencing Data ◽

Smrt Sequencing ◽

Short Read ◽

Short Read Sequencing ◽

Sequencing Method ◽

Long Read ◽

S Allele

The SLC6A4 gene has been implicated in psychiatric disorder susceptibility and antidepressant response variability. The SLC6A4 promoter is defined by a variable number of homologous 20–24 bp repeats (5-HTTLPR), and long (L) and short (S) alleles are associated with higher and lower expression, respectively. However, this insertion/deletion variant is most informative when considered as a haplotype with the rs25531 and rs25532 variants. Therefore, we developed a long-read single molecule real-time (SMRT) sequencing method to interrogate the SLC6A4 promoter region. A total of 120 samples were subjected to SLC6A4 long-read SMRT sequencing, primarily selected based on available short-read sequencing data. Short-read genome sequencing from the 1000 Genomes (1KG) Project (~5X) and the Genetic Testing Reference Material Coordination Program (~45X), as well as high-depth short-read capture-based sequencing (~330X), could not identify the 5-HTTLPR short (S) allele, nor could short-read sequencing phase any identified variants. In contrast, long-read SMRT sequencing unambiguously identified the 5-HTTLPR short (S) allele (frequency of 0.467) and phased SLC6A4 promoter haplotypes. Additionally, discordant rs25531 genotypes were reviewed and determined to be short-read errors. Taken together, long-read SMRT sequencing is an innovative and robust method for phased resolution of the SLC6A4 promoter, which could enable more accurate pharmacogenetic testing for both research and clinical applications.

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A high-throughput multiplexing and selection strategy to complete bacterial genomes

GigaScience ◽

10.1093/gigascience/giab079 ◽

2021 ◽

Vol 10 (12) ◽

Cited By ~ 1

Author(s):

Sergio Arredondo-Alonso ◽

Anna K Pöntinen ◽

François Cléon ◽

Rebecca A Gladstone ◽

Anita C Schürch ◽

...

Keyword(s):

High Throughput ◽

Selection Strategy ◽

Bacterial Isolates ◽

Sequencing Data ◽

Bacterial Genomes ◽

Short Read ◽

Short Read Sequencing ◽

Long Read ◽

Isolate Selection ◽

Selection Of

Abstract Background Bacterial whole-genome sequencing based on short-read technologies often results in a draft assembly formed by contiguous sequences. The introduction of long-read sequencing technologies permits those contiguous sequences to be unambiguously bridged into complete genomes. However, the elevated costs associated with long-read sequencing frequently limit the number of bacterial isolates that can be long-read sequenced. Here we evaluated the recently released 96 barcoding kit from Oxford Nanopore Technologies (ONT) to generate complete genomes on a high-throughput basis. In addition, we propose an isolate selection strategy that optimizes a representative selection of isolates for long-read sequencing considering as input large-scale bacterial collections. Results Despite an uneven distribution of long reads per barcode, near-complete chromosomal sequences (assembly contiguity = 0.89) were generated for 96 Escherichia coli isolates with associated short-read sequencing data. The assembly contiguity of the plasmid replicons was even higher (0.98), which indicated the suitability of the multiplexing strategy for studies focused on resolving plasmid sequences. We benchmarked hybrid and ONT-only assemblies and showed that the combination of ONT sequencing data with short-read sequencing data is still highly desirable (i) to perform an unbiased selection of isolates for long-read sequencing, (ii) to achieve an optimal genome accuracy and completeness, and (iii) to include small plasmids underrepresented in the ONT library. Conclusions The proposed long-read isolate selection ensures the completion of bacterial genomes that span the genome diversity inherent in large collections of bacterial isolates. We show the potential of using this multiplexing approach to close bacterial genomes on a high-throughput basis.

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A high-throughput multiplexing and selection strategy to complete bacterial genomes

10.1101/2021.06.14.448320 ◽

2021 ◽

Author(s):

Sergio Arredondo-Alonso ◽

Anna K. Pöntinen ◽

François Cléon ◽

Rebecca A. Gladstone ◽

Anita C. Schürch ◽

...

Keyword(s):

High Throughput ◽

Selection Strategy ◽

Bacterial Isolates ◽

Sequencing Data ◽

Bacterial Genomes ◽

Short Read ◽

Short Read Sequencing ◽

Long Read ◽

Isolate Selection ◽

Selection Of

Background: Bacterial whole-genome sequencing based on short-read sequencing data often results in a draft assembly formed by contiguous sequences. The introduction of long-read sequencing technologies permits to unambiguously bridge those contiguous sequences into complete genomes. However, the elevated costs associated with long-read sequencing frequently limit the number of bacterial isolates that can be long-read sequenced. Here we evaluated the recently released 96 barcoding kit from Oxford Nanopore Technologies (ONT) to generate complete genomes on a high-throughput basis. In addition, we propose a long-read isolate selection strategy that optimizes a representative selection of isolates from large-scale bacterial collections. Results: Despite an uneven distribution of long-reads per barcode, near-complete chromosomal sequences (assembly contiguity = 0.89) were generated for 96 Escherichia coli isolates with associated short-read sequencing data. The assembly contiguity of the plasmid replicons was even higher (0.98) which indicated the suitability of the multiplexing strategy for studies focused on resolving plasmid sequences. We benchmarked hybrid and ONT-only assemblies and showed that the combination of ONT sequencing data with short-read sequencing data is still highly desirable: (i) to perform an unbiased selection of isolates for long-read sequencing, (ii) to achieve an optimal genome accuracy and completeness, and (iii) to include small plasmids underrepresented in the ONT library. Conclusions: The proposed long-read isolate selection ensures completing bacterial genomes of isolates that span the genome diversity inherent in large collections of bacterial isolates. We show the potential of using this multiplexing approach to close bacterial genomes on a high-throughput basis.

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A Single-Molecule Long-Read Survey of Human Transcriptomes using LoopSeq Synthetic Long Read Sequencing

10.1101/532135 ◽

2019 ◽

Cited By ~ 5

Author(s):

Indira Wu ◽

Tuval Ben-Yehezkel

Keyword(s):

Single Molecule ◽

Transcriptome Sequencing ◽

Splice Variants ◽

Error Rates ◽

Full Length ◽

Tissue Samples ◽

Short Read ◽

Short Read Sequencing ◽

Long Read ◽

Sequence Reconstruction

AbstractState-of-the-art short-read transcriptome sequencing methods employ unique molecular identifier (UMI) to accurately classify and count mRNA transcripts. A fundamental limitation of UMI-based short-read transcriptome sequencing is that each read typically covers a small fraction of the transcript sequence. Efforts to accurately characterize splicing isoforms, arguably the largest source of variation in Human gene expression, using short read sequencing have therefore largely relied on computational predictions of transcript isoforms based on indirect observations. Here we describe a transcript counting, synthetic long read method for sequencing whole transcriptomes using short read sequencing platforms and no additional hardware. The method enables full-length mRNA sequence reconstruction at single-nucleotide resolutions with high-throughput, low error rates and UMI based transcript counting using any Illumina sequencer. We describe results from whole transcriptome sequencing from total RNA extracted from 3 human tissue samples: brain, liver, and blood. Reconstructed transcript sequences are characterized and annotated using SQANTI, an analysis pipeline for assessing the sequence quality of long-read transcriptomes. Our results demonstrate that LoopSeq synthetic long-read sequencing can reconstruct contigs up to 3,900nt full-length transcripts using tissue extracted RNA, as well as identify novel splice variants of known junction donors and acceptors.

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Biosynthetic potential of uncultured Antarctic soil bacteria revealed through long-read metagenomic sequencing

The ISME Journal ◽

10.1038/s41396-021-01052-3 ◽

2021 ◽

Author(s):

Valentin Waschulin ◽

Chiara Borsetto ◽

Robert James ◽

Kevin K. Newsham ◽

Stefano Donadio ◽

...

Keyword(s):

Genome Mining ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Full Length ◽

Metagenomic Sequencing ◽

Short Read ◽

Short Read Sequencing ◽

Rich Diversity ◽

Long Read ◽

The Rich

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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The long march: a sample preparation technique that enhances contig length and coverage by high-throughput short-read sequencing

SciVee ◽

10.4016/10180.01 ◽

2009 ◽

Keyword(s):

Sample Preparation ◽

High Throughput ◽

Preparation Technique ◽

Short Read ◽

Contig Length ◽

Short Read Sequencing ◽

Sample Preparation Technique

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Long-range single-molecule mapping of chromatin modification in eukaryotes

10.1101/2021.07.08.451578 ◽

2021 ◽

Author(s):

Zhe Weng ◽

Fengying Ruan ◽

Weitian Chen ◽

Zhe Xie ◽

Yeming Xie ◽

...

Keyword(s):

Histone Modification ◽

Long Range ◽

Single Molecule ◽

Protein A ◽

Chromatin Modification ◽

Protein Binding Sites ◽

Short Read Sequencing ◽

Third Generation Sequencing ◽

Long Read ◽

Generation Sequencing

The epigenetic modifications of histones are essential marks related to the development and disease pathogenesis, including human cancers. Mapping histone modification has emerged as the widely used tool for studying epigenetic regulation. However, existing approaches limited by fragmentation and short-read sequencing cannot provide information about the long-range chromatin states and represent the average chromatin status in samples. We leveraged the advantage of long read sequencing to develop a method "BIND&MODIFY" for profiling the histone modification of individual DNA fiber. Our approach is based on the recombinant fused protein A-EcoGII, which tethers the methyltransferase EcoGII to the protein binding sites and locally labels the neighboring DNA regions through artificial methylations. We demonstrate that the aggregated BIND&MODIFY signal matches the bulk-level ChIP-seq and CUT&TAG, observe the single-molecule heterogenous histone modification status, and quantify the correlation between distal elements. This method could be an essential tool in the future third-generation sequencing ages.

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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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