Full-length isoform concatenation sequencing to resolve cancer transcriptome complexity.

Cancers exhibit complex transcriptomes with aberrant splicing that induces isoform-level differential expression compared to non-diseased tissues. Transcriptomic profiling using short-read sequencing has utility in providing a cost-effective approach for evaluating isoform expression, although short-read assembly displays limitations in the accurate inference of full-length transcripts. Long-read RNA sequencing (Iso-Seq), using the Pacific Biosciences (PacBio) platform, can overcome such limitations by providing full-length isoform sequence resolution which requires no read assembly and represents native expressed transcripts. A constraint of the Iso-Seq protocol is due to fewer reads output per instrument run, which, as an example, can consequently affect the detection of lowly expressed transcripts. To address these deficiencies, we developed a concatenation workflow, PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq), designed to increase the number of unique, sequenced PacBio long-reads thereby improving overall detection of unique isoforms. In addition, we anticipate that the increase in read depth will help improve the detection of moderate to low-level expressed isoforms. In sequencing a commercial reference (Spike-In RNA Variants; SIRV) with known isoform complexity we demonstrated a 3.4-fold increase in read output per run and improved SIRV recall when using the PB_FLIC-Seq method compared to the same samples processed with the Iso-Seq protocol. We applied this protocol to a translational cancer case, also demonstrating the utility of the PB_FLIC-Seq method for identifying differential full-length isoform expression in a pediatric diffuse midline glioma compared to its adjacent non-malignant tissue. Our data analysis revealed increased expression of extracellular matrix (ECM) genes within the tumor sample, including an isoform of the Secreted Protein Acidic and Cysteine Rich (SPARC) gene that was expressed 11,676-fold higher than in the adjacent non-malignant tissue. Finally, by using the PB_FLIC-Seq method, we detected several cancer-specific novel isoforms. This work describes a concatenation-based methodology for increasing the number of sequenced full-length isoform reads on the PacBio platform, yielding improved discovery of expressed isoforms. We applied this workflow to profile the transcriptome of a pediatric diffuse midline glioma and adjacent non-malignant tissue. Our findings of cancer-specific novel isoform expression further highlight the importance of long-read sequencing for characterization of complex tumor transcriptomes.

Wijeratne S ，Gonzalez MEH ，Roach K ，Miller KE ，Schieffer KM ，Fitch JR ，Leonard J ，White P ，Kelly BJ ，Cottrell CE ，Mardis ER ，Wilson RK ，Miller AR ... -  《BMC GENOMICS》

Pacific Biosciences Fusion and Long Isoform Pipeline for Cancer Transcriptome-Based Resolution of Isoform Complexity.

Genomic profiling using short-read sequencing has utility in detecting disease-associated variation in both DNA and RNA. However, given the frequent occurrence of structural variation in cancer, molecular profiling using long-read sequencing improves the resolution of such events. For example, the Pacific Biosciences long-read RNA-sequencing (Iso-Seq) transcriptome protocol provides full-length isoform characterization, discernment of allelic phasing, and isoform discovery, and identifies expressed fusion partners. The Pacific Biosciences Fusion and Long Isoform Pipeline (PB_FLIP) incorporates a suite of RNA-sequencing software analysis tools and scripts to identify expressed fusion partners and isoforms. In addition, sequencing of a commercial reference (Spike-In RNA Variants) with known isoform complexity was performed and demonstrated high recall of the Iso-Seq and PB_FLIP workflow to benchmark our protocol and analysis performance. This study describes the utility of Iso-Seq and PB_FLIP analysis in improving deconvolution of complex structural variants and isoform detection within an institutional pediatric and adolescent/young adult translational cancer research cohort. The exemplar case studies demonstrate that Iso-Seq and PB_FLIP discover novel expressed fusion partners, resolve complex intragenic alterations, and discriminate between allele-specific expression profiles.

Miller AR ，Wijeratne S ，McGrath SD ，Schieffer KM ，Miller KE ，Lee K ，Mathew M ，LaHaye S ，Fitch JR ，Kelly BJ ，White P ，Mardis ER ，Wilson RK ，Cottrell CE ，Magrini V ... -  《-》

A survey of the complex transcriptome from the highly polyploid sugarcane genome using full-length isoform sequencing and de novo assembly from short read sequencing.

Hoang NV ，Furtado A ，Mason PJ ，Marquardt A ，Kasirajan L ，Thirugnanasambandam PP ，Botha FC ，Henry RJ ... -  《BMC GENOMICS》

High-throughput RNA isoform sequencing using programmed cDNA concatenation.

Full-length RNA-sequencing methods using long-read technologies can capture complete transcript isoforms, but their throughput is limited. We introduce multiplexed arrays isoform sequencing (MAS-ISO-seq), a technique for programmably concatenating complementary DNAs (cDNAs) into molecules optimal for long-read sequencing, increasing the throughput >15-fold to nearly 40 million cDNA reads per run on the Sequel IIe sequencer. When applied to single-cell RNA sequencing of tumor-infiltrating T cells, MAS-ISO-seq demonstrated a 12- to 32-fold increase in the discovery of differentially spliced genes.

Al'Khafaji AM ，Smith JT ，Garimella KV ，Babadi M ，Popic V ，Sade-Feldman M ，Gatzen M ，Sarkizova S ，Schwartz MA ，Blaum EM ，Day A ，Costello M ，Bowers T ，Gabriel S ，Banks E ，Philippakis AA ，Boland GM ，Blainey PC ，Hacohen N ... -  《-》

Transcript Profiling Using Long-Read Sequencing Technologies.

Bayega A ，Wang YC ，Oikonomopoulos S ，Djambazian H ，Fahiminiya S ，Ragoussis J ... -  《-》