Alison D. Tang

While splicing changes caused by somatic mutations in SF3B1 are known, identifying full-length isoform changes may better elucidate the functional consequences of these mutations. We report nanopore sequencing of full-length cDNA from CLL samples with and without SF3B1 mutation, as well as normal B cell samples, giving a total of 149 million pass reads. We present FLAIR (Full-Length Alternative Isoform analysis of RNA), a computational workflow to identify high-confidence transcripts, perform differential splicing event analysis, and differential isoform analysis. Using nanopore reads, we demonstrate differential 3' splice site changes associated with SF3B1 mutation, agreeing with previous studies. We also observe a strong downregulation of intron retention events associated with SF3B1 mutation. Full-length transcript analysis links multiple alternative splicing events together and allows for better estimates of the abundance of productive versus unproductive isoforms. Our work demonstrates the potential utility of nanopore sequencing for cancer and splicing research.

The Drosophila Genome Nexus is a population genomic resource that provides D. melanogaster genomes from multiple sources. To facilitate comparisons across data sets, genomes are aligned using a common reference alignment pipeline which involves two rounds of mapping. Regions of residual heterozygosity, identity-by-descent, and recent population admixture are annotated to enable data filtering based on the user's needs. Here, we present a significant expansion of the Drosophila Genome Nexus, which brings the current data object to a total of 1,121 wild-derived genomes. New additions include 305 previously unpublished genomes from inbred lines representing six population samples in Egypt, Ethiopia, France, and South Africa, along with another 193 genomes added from recently-published data sets. We also provide an aligned D. simulans genome to facilitate divergence comparisons. This improved resource will broaden the range of population genomic questions that can addressed from multi-population allele frequencies and haplotypes in this model species. The larger set of genomes will also enhance the discovery of functionally relevant natural variation that exists within and between populations.

The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

ABSTRACT High throughput cDNA sequencing technologies have dramatically advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and because modifications are not carried forward in cDNA. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study focused on poly(A) RNA from the human cell line GM12878, generating 9.9 million aligned sequence reads. These native RNA reads had an aligned N50 length of 1294 bases, and a maximum aligned length of over 21,000 bases. A total of 78,199 high-confidence isoforms were identified by combining long nanopore reads with short higher accuracy Illumina reads. We describe strategies for assessing 3′ poly(A) tail length, base modifications and transcript haplotypes from nanopore RNA data. Together, these nanopore-based techniques are poised to deliver new insights into RNA biology. DISCLOSURES MA holds shares in Oxford Nanopore Technologies (ONT). MA is a paid consultant to ONT. REW, WT, TG, JRT, JQ, NJL, JTS, NS, AB, MA, HEO, MJ, and ML received reimbursement for travel, accommodation and conference fees to speak at events organised by ONT. NL has received an honorarium to speak at an ONT company meeting. WT has two patents (8,748,091 and 8,394,584) licensed to Oxford Nanopore. JTS, ML and MA received research funding from ONT.