Detecting haplotype-specific transcript variation in long reads with FLAIR2.

Background: RNA-seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants (SNVs) in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme that mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung adenocarcinoma cells and associated with splicing regulation.

Identifying and quantifying isoforms from accurate full-length transcriptome sequencing reads with Mandalorion.

In this manuscript, we introduce and benchmark Mandalorion v4.1 for the identification and quantification of full-length transcriptome sequencing reads. It further improves upon the already strong performance of Mandalorion v3.

Detecting haplotype-specific transcript variation in long reads with FLAIR2.

Background: RNA-Seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme which mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung ADC cells and associated with splicing regulation.

Single-cell isoform analysis in human immune cells.

High-throughput single-cell analysis today is facilitated by protocols like the 10X Genomics platform or Drop-Seq which generate cDNA pools in which the origin of a transcript is encoded at its 5' or 3' end. Here, we used R2C2 to sequence and demultiplex 12 million full-length cDNA molecules generated by the 10X Genomics platform from ~3000 peripheral blood mononuclear cells. We use these reads, independent from Illumina data, to identify B cell, T cell, and monocyte clusters and generate isoform-level transcriptomes for cells and cell types.

Realizing the potential of full-length transcriptome sequencing.

Long-read sequencing holds great potential for transcriptome analysis because it offers researchers an affordable method to annotate the transcriptomes of non-model organisms. This, in turn, will greatly benefit future work on less-researched organisms like unicellular eukaryotes that cannot rely on large consortia to generate these transcriptome annotations. However, to realize this potential, several remaining molecular and computational challenges will have to be overcome.

Depletion of Hemoglobin Transcripts and Long-Read Sequencing Improves the Transcriptome Annotation of the Polar Bear ().

Transcriptome studies evaluating whole blood and tissues are often confounded by overrepresentation of highly abundant transcripts. These abundant transcripts are problematic, as they compete with and prevent the detection of rare RNA transcripts, obscuring their biological importance. This issue is more pronounced when using long-read sequencing technologies for isoform-level transcriptome analysis, as they have relatively lower throughput compared to short-read sequencers.

Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA.

High-throughput short-read sequencing has revolutionized how transcriptomes are quantified and annotated. However, while Illumina short-read sequencers can be used to analyze entire transcriptomes down to the level of individual splicing events with great accuracy, they fall short of analyzing how these individual events are combined into complete RNA transcript isoforms. Because of this shortfall, long-distance information is required to complement short-read sequencing to analyze transcriptomes on the level of full-length RNA transcript isoforms.

Highly Accurate Sequencing of Full-Length Immune Repertoire Amplicons Using Tn5-Enabled and Molecular Identifier-Guided Amplicon Assembly.

Ab repertoire sequencing is a powerful tool to analyze the adaptive immune system. To sequence entire Ab repertoires, amplicons are created from Ab H chain (IgH) transcripts and sequenced on a high-throughput sequencer. The field of immune repertoire sequencing is growing rapidly and the protocols used are steadily improving; however, thus far, immune repertoire sequencing protocols have not been able to sequence full-length immune repertoires including the entire IgH V region and enough of the IgH C region to identify isotype subtypes.