Defining a personal, allele-specific, and single-molecule long-read transcriptome

Abstract

Personal transcriptomes in which all of an individual's genetic variants (e.g., single nucleotide variants) and transcript isoforms (transcription start sites, splice sites, and polyA sites) are defined and quantified for full-length transcripts are expected to be important for understanding individual biology and disease, but have not been described previously. To obtain such transcriptomes, we sequenced the lymphoblastoid transcriptomes of three family members (GM12878 and the parents GM12891 and GM12892) by using a Pacific Biosciences long-read approach complemented with Illumina 101-bp sequencing and made the following observations. First, we found that reads representing all splice sites of a transcript are evident for most sufficiently expressed genes ≤3 kb and often for genes longer than that. Second, we added and quantified previously unidentified splicing isoforms to an existing annotation, thus creating the first personalized annotation to our knowledge. Third, we determined SNVs in a de novo manner and connected them to RNA haplotypes, including HLA haplotypes, thereby assigning single full-length RNA molecules to their transcribed allele, and demonstrated Mendelian inheritance of RNA molecules. Fourth, we show how RNA molecules can be linked to personal variants on a one-by-one basis, which allows us to assess differential allelic expression (DAE) and differential allelic isoforms (DAI) from the phased full-length isoform reads. The DAI method is largely independent of the distance between exon and SNV-in contrast to fragmentation-based methods. Overall, in addition to improving eukaryotic transcriptome annotation, these results describe, to our knowledge, the first large-scale and full-length personal transcriptome.personalized medicine | isoform sequencing | platform comparison | alternative splicing | allele-specific expression S hort-read RNA sequencing (1-7) is a widely used tool in modern day biology. In mammalian transcriptomes, multiintron genes are common and the detection and quantification of different transcript isoforms is of high importance. Recent work has shown that reconstruction and quantification of transcript isoforms from short-read sequencing is insufficiently accurate (8, 9). Simultaneously, a number of research groups have pursued long-read sequencing (8,(10)(11)(12), and such datasets generally excel at connecting different exons, up to entire transcripts, with a compromise of lower sequencing depth. However, these studies have not investigated allelic variants. Such information is crucial for understanding personal transcriptomes and their potential biological consequences.Here, we use the Pacific Biosciences (13) platform ("PacBio") to produce a single-molecule RNA-seq dataset in the GM12878 cell line that is more comprehensive in both length and depth than a recently described dataset from a human organ panel (HOP) (11). We additionally sequenced cDNA from the same cell line by using 101-bp paired-end (PE) sequencing on the Illumina platform, to show the properties of gene...

The dynamic landscape of fission yeast meiosis alternative-splice isoforms

“…The average length of CCS reads was 1285 bp, which was slightly longer than previous reports ( Fig. 2B; Sharon et al 2013;Thomas et al 2014;Tilgner et al 2014). The average length of FL CCS reads and Iso-Seq reads were 1094 and 1178 bp, respectively, and the length distributions did not vary across time points (Supplemental Fig.…”

“…PacBio sequencing features very long reads, which are suitable for full-length cDNA sequencing. Recently, PacBio sequencing has been used to examine the transcriptome in multiple metazoan organisms, such as chicken and human, and multiple plants, and identified thousands of annotated and novel isoforms (Au et al 2013;Sharon et al 2013;Martin et al 2014;Thomas et al 2014;Tilgner et al 2014;Dong et al 2015;Minoche et al 2015). To systematically characterize isoforms and explore their differential regulations in fission yeast, we performed time-course isoform-level profiling during the meiosis of S. pombe.…”

Advancements in Next-Generation Sequencing

“…Pacific Biosciences launched its SMRT Analysis 2.2 software with support for the Iso-seq method of analyzing full-length transcripts and gene isoforms, without the requirement of assembly. Several studies have utilized this approach, including analyses of human pluripotent stem cells (11), allele-specific transcription (116), and alternative splicing (118). It will be interesting to see how the transcriptional analysis field evolves as long-read sequencing becomes more available and cost effective, particularly in light of a study that raised questions about errors in short-read data and transcriptional analysis (99).…”

Direct chromosome-length haplotyping by single-cell sequencing

“…Accession numbers are SRR1163655 (NA12878), SRR1163657 (NA12891), and SRR1163658 (NA12892) (Tilgner et al 2014). …”