RNA-seq Pipeline with Fusion Detection

RNA-seq was performed using TruSeq library preparation and HiSeq 2000 and 2500 sequencers (Illumina). All sequence reads were paired-end, and was performed using (1) total RNA and stranded RNA-seq [75 or 100 base-pair (bp) reads]; (2) polyA-selected mRNA (50, 75 or 100bp reads). Sequencing reads were mapped to the GRCh37 human genome reference by STAR^{44 (link)} (version 2.4.2a) through the suggested two-pass mapping pipeline. Gene annotation downloaded from Ensembl website (see URLs) was used for STAR mapping and the following read-count evaluation. All the samples were sequenced with RefSeq coding region covered with 30-fold coverage ≥15% (median ± standard deviation, 37.2±7.5%). CICERO^{5 (link)} and FusionCatcher^{45 ,46 (link)} were used to detect fusions, and all the reported rearrangements were manually reviewed to keep the reliable ones. Due to the complexity of DUX4 rearrangements, some of the DUX4 fusions were manually rescued by checking the aligned reads within IGV browser^{47 (link)}. RNA extracted from flow sorted normal B lymphoid cells were used for RNA-seq and details were provided in our previous study⁴⁸.

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Gu Z., Churchman M.L., Roberts K.G., Moore I., Zhou X., Nakitandwe J., Hagiwara K., Pelletier S., Gingras S., Berns H., Payne-Turner D., Hill A., Iacobucci I., Shi L., Pounds S., Cheng C., Pei D., Qu C., Newman S., Devidas M., Dai Y., Reshmi S.C., Gastier-Foster J., Raetz E.A., Borowitz M.J., Wood B.L., Carroll W.L., Zweidler-McKay P.A., Rabin K.R., Mattano L.A., Maloney K.W., Rambaldi A., Spinelli O., Radich J.P., Minden M.D., Rowe J.M., Luger S., Litzow M.R., Tallman M.S., Racevskis J., Zhang Y., Bhatia R., Kohlschmidt J., Mrózek K., Bloomfield C.D., Stock W., Kornblau S., Kantarjian H.M., Konopleva M., Evans W., Jeha S., Pui C.H., Yang J., Paietta E., Downing J., Relling M.V., Zhang J., Loh M.L., Hunger S.P, & Mullighan C.G. (2019). PAX5-driven Subtypes of B-progenitor Acute Lymphoblastic Leukemia. Nature genetics, 51(2), 296-307.

Publication 2019

HiSeq 2000 and 2500 sequencers

Base pair Dux4 Gene annotation Human Human genome Library Lymphoid cells Polya mrna Rearrangements Rna seq

Corresponding Organization :

Other organizations : St. Jude Children's Research Hospital, University of Pittsburgh, University of Florida, Nationwide Children's Hospital, New York University, Johns Hopkins University, University of Washington, NYU Langone Health, ImmunoGen (United States), Baylor College of Medicine, Children's Hospital Colorado, University of Colorado Denver, Ospedale Papa Giovanni XXIII, Fred Hutch Cancer Center, Princess Margaret Cancer Centre, University Health Network, Shaare Zedek Medical Center, University of Pennsylvania, Mayo Clinic in Arizona, Memorial Sloan Kettering Cancer Center, Montefiore Medical Center, Albert Einstein College of Medicine, University of Alabama at Birmingham, The Ohio State University, University of Chicago Medical Center, The University of Texas MD Anderson Cancer Center, UCSF Benioff Children's Hospital, Children's Hospital of Philadelphia

Top 5 similar protocols

Protocol cited in 16 other protocols

Variable analysis

independent variables

TruSeq library preparation
HiSeq 2000 and 2500 sequencers (Illumina)
Total RNA and stranded RNA-seq [75 or 100 base-pair (bp) reads]
PolyA-selected mRNA (50, 75 or 100bp reads)

dependent variables

Gene expression levels

control variables

GRCh37 human genome reference
Ensembl gene annotation
RefSeq coding region coverage ≥15% (median ± standard deviation, 37.2±7.5%)
RNA extracted from flow sorted normal B lymphoid cells

Annotations

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