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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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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Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution

BMC Microbiology ◽

10.1186/s12866-021-02094-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yoshiyuki Matsuo ◽

Shinnosuke Komiya ◽

Yoshiaki Yasumizu ◽

Yuki Yasuoka ◽

Katsura Mizushima ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Amplicon Sequencing ◽

Species Level ◽

Full Length ◽

Rrna Gene ◽

Short Read ◽

Short Read Sequencing ◽

Long Read

Abstract Background Species-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples. Results We modified our existing protocol for full-length 16S rRNA gene amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S rRNA gene amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition. Conclusions Our present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene.

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scCAT-seq:single-cell identification and quantification of mRNA isoforms by cost-effective short-read sequencing of cap and tail

10.1101/2019.12.11.873505 ◽

2019 ◽

Author(s):

Youjin Hu ◽

Jiawei Zhong ◽

Yuhua Xiao ◽

Zheng Xing ◽

Katherine Sheu ◽

...

Keyword(s):

Single Cell ◽

Learning Algorithm ◽

Single Cells ◽

Full Length ◽

Translation Efficiency ◽

Mrna Isoforms ◽

Short Read ◽

Short Read Sequencing ◽

Long Read ◽

Identification And Quantification

AbstractThe differences in transcription start sites (TSS) and transcription end sites (TES) among gene isoforms can affect the stability, localization, and translation efficiency of mRNA. Isoforms also allow a single gene different functions across various tissues and cells However, methods for efficient genome-wide identification and quantification of RNA isoforms in single cells are still lacking. Here, we introduce single cell Cap And Tail sequencing (scCAT-seq). In conjunction with a novel machine learning algorithm developed for TSS/TES characterization, scCAT-seq can demarcate transcript boundaries of RNA transcripts, providing an unprecedented way to identify and quantify single-cell full-length RNA isoforms based on short-read sequencing. Compared with existing long-read sequencing methods, scCAT-seq has higher efficiency with lower cost. Using scCAT-seq, we identified hundreds of previously uncharacterized full-length transcripts and thousands of alternative transcripts for known genes, quantitatively revealed cell-type specific isoforms with alternative TSSs/TESs in dorsal root ganglion (DRG) neurons, mature oocytes and ageing oocytes, and generated the first atlas of the non-human primate cornea. The approach described here can be widely adapted to other short-read or long-read methods to improve accuracy and efficiency in assessing RNA isoform dynamics among single cells.

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Emerging Techniques for the Characterization of Nano-Mechanical Properties of Integrated Circuit Components

ISTFA 2008: Conference Proceedings from the 34th International Symposium for Testing and Failure Analysis ◽

10.31399/asm.cp.istfa2008p0121 ◽

2008 ◽

Author(s):

Nicholas Randall ◽

Rahul Premachandran Nair

Keyword(s):

Mechanical Properties ◽

Quality Control ◽

Integrated Circuits ◽

Integrated Circuit ◽

Manufacturing Process ◽

Solder Bumps ◽

Initial Work ◽

Circuit Components ◽

Materials Used

Abstract With the growing complexity of integrated circuits (IC) comes the issue of quality control during the manufacturing process. In order to avoid late realization of design flaws which could be very expensive, the characterization of the mechanical properties of the IC components needs to be carried out in a more efficient and standardized manner. The effects of changes in the manufacturing process and materials used on the functioning and reliability of the final device also need to be addressed. Initial work on accurately determining several key mechanical properties of bonding pads, solder bumps and coatings using a combination of different methods and equipment has been summarized.

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