Deep RNA sequencing analysis of readthrough gene fusions in human prostate adenocarcinoma and reference samples.
BACKGROUND
Readthrough fusions across adjacent genes in the genome, or transcription-induced chimeras (TICs), have been estimated using expressed sequence tag (EST) libraries to involve 4-6% of all genes. Deep transcriptional sequencing (RNA-Seq) now makes it possible to study the occurrence and expression levels of TICs in individual samples across the genome.


METHODS
We performed single-end RNA-Seq on three human prostate adenocarcinoma samples and their corresponding normal tissues, as well as brain and universal reference samples. We developed two bioinformatics methods to specifically identify TIC events: a targeted alignment method using artificial exon-exon junctions within 200,000 bp from adjacent genes, and genomic alignment allowing splicing within individual reads. We performed further experimental verification and characterization of selected TIC and fusion events using quantitative RT-PCR and comparative genomic hybridization microarrays.


RESULTS
Targeted alignment against artificial exon-exon junctions yielded 339 distinct TIC events, including 32 gene pairs with multiple isoforms. The false discovery rate was estimated to be 1.5%. Spliced alignment to the genome was less sensitive, finding only 18% of those found by targeted alignment in 33-nt reads and 59% of those in 50-nt reads. However, spliced alignment revealed 30 cases of TICs with intervening exons, in addition to distant inversions, scrambled genes, and translocations. Our findings increase the catalog of observed TIC gene pairs by 66%.We verified 6 of 6 predicted TICs in all prostate samples, and 2 of 5 predicted novel distant gene fusions, both private events among 54 prostate tumor samples tested. Expression of TICs correlates with that of the upstream gene, which can explain the prostate-specific pattern of some TIC events and the restriction of the SLC45A3-ELK4 e4-e2 TIC to ERG-negative prostate samples, as confirmed in 20 matched prostate tumor and normal samples and 9 lung cancer cell lines.


CONCLUSIONS
Deep transcriptional sequencing and analysis with targeted and spliced alignment methods can effectively identify TIC events across the genome in individual tissues. Prostate and reference samples exhibit a wide range of TIC events, involving more genes than estimated previously using ESTs. Tissue specificity of TIC events is correlated with expression patterns of the upstream gene. Some TIC events, such as MSMB-NCOA4, may play functional roles in cancer.
Department of Bioinformatics and Molecular Biology, Genentech, Inc, South San Francisco, California 94080, USA.
Serban
S
Nacu
Wenlin
W
Yuan
Zhengyan
Z
Kan
Deepali
D
Bhatt
Celina Sanchez
CS
Rivers
Jeremy
J
Stinson
Brock A
BA
Peters
Zora
Z
Modrusan
Kenneth
K
Jung
Somasekar
S
Seshagiri
Thomas D
TD
Wu
D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D001483:Base Sequence; D055028:Comparative Genomic Hybridization / Q000379:methods; D030541:Databases, Genetic; D020224:Expressed Sequence Tags; D050939:Gene Fusion; D006801:Humans; D008297:Male; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000473:pathology; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017423:Sequence Analysis, RNA / Q000379:methods*
J Biol Chem. 2003 Sep 5;278(36):34035-41
12824192
Proc Natl Acad Sci U S A. 2009 Jul 28;106(30):12353-8
19592507
Nat Genet. 2008 Mar;40(3):310-5
18264096
Proteins. 1997 Jul;28(3):405-20
9223186
J Biol Chem. 2003 May 30;278(22):19619-26
12649293
Genome Res. 2000 Nov;10(11):1743-56
11076860
Science. 2008 Sep 5;321(5894):1357-61
18772439
J Biol Chem. 2001 May 11;276(19):16561-6
11278528
Nat Biotechnol. 2009 Nov;27(11):1005-11
19881495
J Biol Chem. 1998 Jun 26;273(26):16005-10
9632650
Nat Genet. 2008 Dec;40(12):1413-5
18978789
Genomics. 1999 May 1;57(3):371-9
10329003
Nucleic Acids Res. 2002 Jan 1;30(1):207-10
11752295
Science. 2008 Aug 15;321(5891):956-60
18599741
Oncogene. 2007 Apr 19;26(18):2667-73
17043636
Genome Biol. 2010;11(10):R104
20964841
Cancer Res. 2009 Apr 1;69(7):2734-8
19293179
Genome Res. 2011 Jan;21(1):56-67
21036922
Genome Res. 2009 Feb;19(2):167-77
19056696
Genome Biol. 2010;11(3):R34
20236510
BMC Genomics. 2007 Oct 01;8:348
17908320
Genes Chromosomes Cancer. 2002 Dec;35(4):311-7
12378525
Genome Res. 2007 Jun;17(6):746-59
17567994
Genome Res. 2006 Jan;16(1):30-6
16344562
Cancer Res. 2006 Sep 1;66(17):8347-51
16951141
Genome Res. 2006 Jan;16(1):37-44
16344564
BMC Genomics. 2006 Jun 13;7:148
16772034
Genome Res. 2005 Jul;15(7):987-97
15998911
Nature. 2008 Nov 27;456(7221):470-6
18978772
Cancer Res. 2004 Nov 1;64(21):7647-54
15520162
Nature. 2009 Mar 5;458(7234):97-101
19136943
Genome Biol. 2009;10(10):R115
19835606
Annu Rev Biochem. 2007;76:51-74
17352659
Cancer Res. 2006 Nov 15;66(22):10658-63
17108102
Nat Methods. 2008 Jul;5(7):613-9
18516046
Nat Rev Genet. 2009 Jan;10(1):57-63
19015660
Nature. 2009 Apr 9;458(7239):719-24
19360079
Biochem Biophys Res Commun. 2006 Dec 1;350(4):890-9
17045569
Nat Biotechnol. 2006 Sep;24(9):1115-22
16964225
Bioinformatics. 2009 May 1;25(9):1105-11
19289445
EMBO J. 2002 Nov 1;21(21):5711-20
12411489
Bioinformatics. 2010 Apr 1;26(7):873-81
20147302
Genome Res. 2007 Jun;17(6):828-38
17568001
Bioinformatics. 2005 May 1;21(9):1859-75
15728110
J Biol Chem. 2009 Jul 3;284(27):18033-46
19416963
Nat Genet. 2008 Mar;40(3):316-21
18264097
Pathol Oncol Res. 2008 Sep;14(3):243-51
18553159
Proc Natl Acad Sci U S A. 2003 Jan 7;100(1):189-92
12502788
Mol Cancer. 2009 Jan 19;8:5
19152679
Bioinformatics. 2008 Aug 15;24(16):i174-80
18689821
Science. 2005 Oct 28;310(5748):644-8
16254181
Genome Biol. 2006;7 Suppl 1:S12.1-14
16925834
Cancer Res. 2010 May 1;70(9):3440-9
20388783
Cell. 2009 Dec 11;139(6):1069-83
19962179
Nat Biotechnol. 2006 Sep;24(9):1151-61
16964229