SQANTI: extensive characterization of long read transcript sequences for quality control in full-length transcriptome identification and quantification

ABSTRACTHigh-throughput sequencing of full-length transcripts using long reads has paved the way for the discovery of thousands of novel transcripts, even in very well annotated organisms as mice and humans. Nonetheless, there is a need for studies and tools that characterize these novel isoforms. Here we present SQANTI, an automated pipeline for the classification of long-read transcripts that computes 47 descriptors that can be used to assess the quality of the data and of the preprocessing pipelines. We applied SQANTI to a neuronal mouse transcriptome using PacBio long reads and illustrate how the tool is effective in readily describing the composition of and characterizing the full-length transcriptome. We perform extensive evaluation of ToFU PacBio transcripts by PCR to reveal that an important number of the novel transcripts are technical artifacts of the sequencing approach, and that SQANTI quality descriptors can be used to engineer a filtering strategy to remove them. Most novel transcripts in this curated transcriptome are novel combinations of existing splice sites, result more frequently in novel ORFs than novel UTRs and are enriched in both general metabolic and neural specific functions. We show that these new transcripts have a major impact in the correct quantification of transcript levels by state-of-the-art short-read based quantification algorithms. By comparing our iso-transcriptome with public proteomics databases we find that alternative isoforms are elusive to proteogenomics detection and are variable in protein changes with respect to the principal isoform of their genes. SQANTI allows the user to maximize the analytical outcome of long read technologies by providing the tools to deliver quality-evaluated and curated full-length transcriptomes. SQANTI is available at https://bitbucket.org/ConesaLab/sqanti.

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Complete and validated genomes from a metagenome

10.1101/2020.04.08.032540 ◽

2020 ◽

Cited By ~ 1

Author(s):

Daniel J Giguere ◽

Alexander T Bahcheli ◽

Benjamin R Joris ◽

Julie M Paulssen ◽

Lisa M Gieg ◽

...

Keyword(s):

Naphthenic Acid ◽

Short Read ◽

Long Reads ◽

Complete Genomes ◽

Long Read ◽

Genomic Characterization ◽

Metagenomic Assembly ◽

Candidate Phyla Radiation

0.1AbstractThe assembly and binning of metagenomically-assembled genomes (MAGs) using Illumina sequencing has improved the genomic characterization of unculturable communities. However, short-read-only metagenomic assemblies rarely result in completed genomes because of the difficulty assembling repetitive regions. Here, we present a strategy to complete and validate multiple MAGs from a bacterial community using a combination of short and ultra long reads (N50 > 25 kb). Our strategy is to perform an initial long read-only metagenomic assembly using metaFlye, followed by multiple rounds of polishing using both long and short reads. To validate the genomes, we verified that longs reads spanned the regions that were not supported by uniquely mapped paired-end Illumina sequences. We obtained multiple complete genomes from a naphthenic acid-degrading community, including one from the recently proposed Candidate Phyla Radiation. The majority of the population is represented by the assembled genomes; recruiting 63.77 % of Nanopore reads, and 64.38 % of Illumina reads. The pipeline we developed will enable researchers to validate genomes from metagenomic assemblies, increasing the quality of metagenomically assembled genomes through additional scrutiny.

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Ultra-accurate microbial amplicon sequencing with synthetic long reads

Microbiome ◽

10.1186/s40168-021-01072-3 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Benjamin J. Callahan ◽

Dmitry Grinevich ◽

Siddhartha Thakur ◽

Michael A. Balamotis ◽

Tuval Ben Yehezkel

Keyword(s):

Microbial Community ◽

16S Rrna ◽

Amplicon Sequencing ◽

Species Level ◽

Full Length ◽

16S Rrna Genes ◽

Rrna Genes ◽

Strain Identification ◽

Long Reads ◽

Long Read

Abstract Background Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. Methods Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads. Results LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens. Conclusions The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.

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Characterization of Full-Length Transcriptome Sequences and Splice Variants of Lateolabrax maculatus by Single-Molecule Long-Read Sequencing and Their Involvement in Salinity Regulation

Frontiers in Genetics ◽

10.3389/fgene.2019.01126 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 4

Author(s):

Yuan Tian ◽

Haishen Wen ◽

Xin Qi ◽

Xiaoyan Zhang ◽

Shikai Liu ◽

...

Keyword(s):

Single Molecule ◽

Splice Variants ◽

Full Length ◽

Long Read ◽

Lateolabrax Maculatus ◽

Transcriptome Sequences

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Characterization of segmental duplications and large inversions using Linked-Reads

10.1101/394528 ◽

2018 ◽

Cited By ~ 4

Author(s):

Fatih Karaoglanoglu ◽

Camir Ricketts ◽

Marzieh Eslami Rasekh ◽

Ezgi Ebren ◽

Iman Hajirasouliha ◽

...

Keyword(s):

High Throughput Sequencing ◽

Segmental Duplications ◽

Sequencing Data ◽

Full Spectrum ◽

Genomic Structural Variation ◽

Split Read ◽

Long Read ◽

Novel Algorithms ◽

Insertion Locus

AbstractMany algorithms aimed at characterizing genomic structural variation (SV) have been developed since the inception of high-throughput sequencing. However, the full spectrum of SVs in the human genome is not yet assessed. Most of the existing methods focus on discovery and genotyping of deletions, insertions, and mobile elements. Detection of balanced SVs with no gain or loss of genomic segments (e.g., inversions) is particularly a challenging task. Long read sequencing has been leveraged to find short inversions but there is still a need to develop methods to detect large genomic inversions. Furthermore, currently there are no algorithms to predict the insertion locus of large interspersed segmental duplications.Here we propose novel algorithms to characterize large (>40Kbp) interspersed segmental duplications and (>80Kbp) inversions using Linked-Read sequencing data. Linked-Read sequencing provides long range information, where Illumina reads are tagged with barcodes that can be used to assign short reads to pools of larger (30-50 Kbp) molecules. Our methods rely on split molecule sequence signature that we have previously described [11]. Similar to the split read, split molecules refer to large segments of DNA that span an SV breakpoint. Therefore, when mapped to the reference genome, the mapping of these segments would be discontinuous. We redesign our earlier algorithm, VALOR, to specifically leverage Linked-Read sequencing data to discover large inversions and characterize interspersed segmental duplications. We implement our new algorithms in a new software package, called VALOR2.AvailabilityVALOR2 is available at https://github.com/BilkentCompGen/valor.

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Comprehensive characterization of single cell full-length isoforms in human and mouse with long-read sequencing

10.1101/2020.08.10.243543 ◽

2020 ◽

Author(s):

Luyi Tian ◽

Jafar S. Jabbari ◽

Rachel Thijssen ◽

Quentin Gouil ◽

Shanika L. Amarasinghe ◽

...

Keyword(s):

Data Integration ◽

Single Cell ◽

Ribosome Biogenesis ◽

Single Cells ◽

Transcript Level ◽

Full Length ◽

Alternative Transcript ◽

Long Read ◽

Comprehensive Characterization

AbstractAlternative splicing shapes the phenotype of cells in development and disease. Long-read RNA-sequencing recovers full-length transcripts but has limited throughput at the single-cell level. Here we developed single-cell full-length transcript sequencing by sampling (FLT-seq), together with the computational pipeline FLAMES to overcome these issues and perform isoform discovery and quantification, splicing analysis and mutation detection in single cells. With FLT-seq and FLAMES, we performed the first comprehensive characterization of the full-length isoform landscape in single cells of different types and species and identified thousands of unannotated isoforms. We found conserved functional modules that were enriched for alternative transcript usage in different cell populations, including ribosome biogenesis and mRNA splicing. Analysis at the transcript-level allowed data integration with scATAC-seq on individual promoters, improved correlation with protein expression data and linked mutations known to confer drug resistance to transcriptome heterogeneity. Our methods reveal previously unseen isoform complexity and provide a better framework for multi-omics data integration.

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Characterization of sintered Cu nanopaste for micro-bumping with Injection Molded Solder technology

International Symposium on Microelectronics ◽

10.4071/2380-4505-2020.1.000113 ◽

2020 ◽

Vol 2020 (1) ◽

pp. 000113-000118

Author(s):

Eiji Nakamura ◽

Toyohiro Aoki ◽

Ryota Yamaguchi ◽

Nobuhiro Sekine ◽

Kuniaki Sueoka ◽

...

Keyword(s):

Cross Sections ◽

Molten Solder ◽

The Novel ◽

Cu Nanoparticles ◽

Shear Testing ◽

Injection Molded ◽

Sintering Conditions ◽

Cu Nanoparticle

Abstract We have previously developed a novel plating-free bumping process using Cu nanopaste and Injection Molded Solder (IMS) technology. In the present study, we investigated the further detail about the microstructural and mechanical properties of sintered Cu nanoparticles formed into a pillar shape. By analyzing cross-sections of Cu nanoparticle pillars sintered in various conditions, we clarified how the sintering conditions affect the microstructural features, including the size and numbers of Cu grains and voids inside sintered Cu nanoparticles. In addition, we conducted the shear testing for the obtained Cu pillars to evaluate relationships between the mechanical strength and the microstructural features. We found that the results of the shear testing were consistent with the microstructural features of the sintered Cu nanoparticles. Finally, we injected molten solder onto the Cu nanoparticle pillars to evaluate the overall feasibility of the developed process. It was confirmed that the molten solder injected by IMS process has good wettability against the sintered Cu nanoparticles, which resulted in the successful bump formation without solder missing. In addition, The IMC layer between the sintered Cu nanoparticles and injected solder was formed well. These results proved the quality of microbumps fabricated by the novel bumping process using Cu nanopaste and IMS.

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ISOdb: A Comprehensive Database of Full-Length Isoforms Generated by Iso-Seq

International Journal of Genomics ◽

10.1155/2018/9207637 ◽

2018 ◽

Vol 2018 ◽

pp. 1-6 ◽

Cited By ~ 1

Author(s):

Shang-Qian Xie ◽

Yue Han ◽

Xiao-Zhou Chen ◽

Tai-Yu Cao ◽

Kai-Kai Ji ◽

...

Keyword(s):

Single Molecule ◽

Full Length ◽

Public Access ◽

Transcript Isoforms ◽

Sequencing Technologies ◽

Long Reads ◽

Depth Analysis ◽

Gene Level ◽

Long Read ◽

Full Length Transcript

The accurate landscape of transcript isoforms plays an important role in the understanding of gene function and gene regulation. However, building complete transcripts is very challenging for short reads generated using next-generation sequencing. Fortunately, isoform sequencing (Iso-Seq) using single-molecule sequencing technologies, such as PacBio SMRT, provides long reads spanning entire transcript isoforms which do not require assembly. Therefore, we have developed ISOdb, a comprehensive resource database for hosting and carrying out an in-depth analysis of Iso-Seq datasets and visualising the full-length transcript isoforms. The current version of ISOdb has collected 93 publicly available Iso-Seq samples from eight species and presents the samples in two levels: (1) sample level, including metainformation, long read distribution, isoform numbers, and alternative splicing (AS) events of each sample; (2) gene level, including the total isoforms, novel isoform number, novel AS number, and isoform visualisation of each gene. In addition, ISOdb provides a user interface in the website for uploading sample information to facilitate the collection and analysis of researchers’ datasets. Currently, ISOdb is the first repository that offers comprehensive resources and convenient public access for hosting, analysing, and visualising Iso-Seq data, which is freely available.

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Overcoming uncollapsed haplotypes in long-read assemblies of non-model organisms

BMC Bioinformatics ◽

10.1186/s12859-021-04118-3 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Nadège Guiglielmoni ◽

Antoine Houtain ◽

Alessandro Derzelle ◽

Karine Van Doninck ◽

Jean-François Flot

Keyword(s):

Genome Assembly ◽

Model Organism ◽

Model Organisms ◽

Post Processing ◽

Diploid Genome ◽

Long Reads ◽

Long Read ◽

Eukaryote Genome ◽

Chromosome Level

Abstract Background Long-read sequencing is revolutionizing genome assembly: as PacBio and Nanopore technologies become more accessible in technicity and in cost, long-read assemblers flourish and are starting to deliver chromosome-level assemblies. However, these long reads are usually error-prone, making the generation of a haploid reference out of a diploid genome a difficult enterprise. Failure to properly collapse haplotypes results in fragmented and structurally incorrect assemblies and wreaks havoc on orthology inference pipelines, yet this serious issue is rarely acknowledged and dealt with in genomic projects, and an independent, comparative benchmark of the capacity of assemblers and post-processing tools to properly collapse or purge haplotypes is still lacking. Results We tested different assembly strategies on the genome of the rotifer Adineta vaga, a non-model organism for which high coverages of both PacBio and Nanopore reads were available. The assemblers we tested (Canu, Flye, NextDenovo, Ra, Raven, Shasta and wtdbg2) exhibited strikingly different behaviors when dealing with highly heterozygous regions, resulting in variable amounts of uncollapsed haplotypes. Filtering reads generally improved haploid assemblies, and we also benchmarked three post-processing tools aimed at detecting and purging uncollapsed haplotypes in long-read assemblies: HaploMerger2, purge_haplotigs and purge_dups. Conclusions We provide a thorough evaluation of popular assemblers on a non-model eukaryote genome with variable levels of heterozygosity. Our study highlights several strategies using pre and post-processing approaches to generate haploid assemblies with high continuity and completeness. This benchmark will help users to improve haploid assemblies of non-model organisms, and evaluate the quality of their own assemblies.

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BiSCoT: Improving large eukaryotic genome assemblies with optical maps

10.1101/674721 ◽

2019 ◽

Cited By ~ 1

Author(s):

Benjamin Istace ◽

Caroline Belser ◽

Jean-Marc Aury

Keyword(s):

Human Genome ◽

Fasta File ◽

Plant Genomes ◽

Link Type ◽

Long Reads ◽

Entire Chromosome ◽

Long Read ◽

Optical Maps ◽

Genome Assemblies

ABSTRACTMotivationLong read sequencing and Bionano Genomics optical maps are two techniques that, when used together, make it possible to reconstruct entire chromosome or chromosome arms structure. However, the existing tools are often too conservative and organization of contigs into scaffolds is not always optimal.ResultsWe developed BiSCoT (Bionano SCaffolding COrrection Tool), a tool that post-processes files generated during a Bionano scaffolding in order to produce an assembly of greater contiguity and quality. BiSCoT was tested on a human genome and four publicly available plant genomes sequenced with Nanopore long reads and improved significantly the contiguity and quality of the assemblies. BiSCoT generates a fasta file of the assembly as well as an AGP file which describes the new organization of the input assembly.AvailabilityBiSCoT and improved assemblies are freely available on Github at http://www.genoscope.cns.fr/biscot and Pypi at https://pypi.org/project/biscot/.

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