From: TopHat: discovering splice junctions with RNA-Seq

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From: TopHat: discovering splice junctions with RNA-Seq Fig. 1. The TopHat pipeline. RNA-Seq reads are mapped against the whole reference genome, and those reads that do not map are set aside. An initial consensus of mapped regions is computed by Maq. Sequences flanking potential donor/acceptor splice sites within neighboring regions are joined to form potential splice junctions. The IUM reads are indexed and aligned to these splice junction sequences. From: TopHat: discovering splice junctions with RNA-Seq Bioinformatics. 2009;25(9):1105-1111. doi:10.1093/bioinformatics/btp120 Bioinformatics | © 2009 The Author(s)This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

From: TopHat: discovering splice junctions with RNA-Seq Fig. 2. An intron entirely overlapped by the 5′-UTR of another transcript. Both isoforms are present in the brain tissue RNA sample. The top track is the normalized uniquely mappable read coverage reported by ERANGE for this region (Mortazavi et al., 2008). The lack of a large coverage gap causes TopHat to report a single island containing both exons. TopHat looks for introns within single islands in order to detect this junction. From: TopHat: discovering splice junctions with RNA-Seq Bioinformatics. 2009;25(9):1105-1111. doi:10.1093/bioinformatics/btp120 Bioinformatics | © 2009 The Author(s)This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

From: TopHat: discovering splice junctions with RNA-Seq Fig. 3. The seed and extend alignment used to match reads to possible splice sites. For each possible splice site, a seed is formed by combining a small amount of sequence upstream of the donor and downstream of the acceptor. This seed, shown in dark gray, is used to query the index of reads that were not initially mapped by Bowtie. Any read containing the seed is checked for a complete alignment to the exons on either side of the possible splice. In the light gray portion of the alignment, TopHat allows a user-specified number of mismatches. Because reads typically contain low-quality base calls on their 3′ ends, TopHat only examines the first 28 bp on the 5′ end of each read by default. From: TopHat: discovering splice junctions with RNA-Seq Bioinformatics. 2009;25(9):1105-1111. doi:10.1093/bioinformatics/btp120 Bioinformatics | © 2009 The Author(s)This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

From: TopHat: discovering splice junctions with RNA-Seq Fig. 4. TopHat sensitivity as RPKM varies. For genes transcribed above 15.0 RPKM, TopHat detects more than 80% reported by ERANGE in the M. musculus brain tissue study. TopHat detects more than 72% of all junctions observed by ERANGE, including those in genes expressed at only a single transcript per cell. A de novo assembly of the RNA-Seq reads, followed by spliced alignment of the assembled transcripts produces markedly poorer sensitivity, detecting around 40% of junctions in genes transcribed above 25.0 RPKM, but comparatively few junctions in more highly transcribed genes. From: TopHat: discovering splice junctions with RNA-Seq Bioinformatics. 2009;25(9):1105-1111. doi:10.1093/bioinformatics/btp120 Bioinformatics | © 2009 The Author(s)This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

From: TopHat: discovering splice junctions with RNA-Seq Fig. 5. The BLAT E-value distribution of known, previously unreported, and randomly generated splice junction sequences when searched against GenBank mouse ESTs. As expected, known junctions have high-quality BLAT hits to the EST database. Randomly-generated junction sequences do not. High-quality BLAT hits for more than 11% of the junctions identified by TopHat suggest that the UCSC gene models for mouse are incomplete. These junctions are almost certainly genuine, and because the mouse EST database is not complete, 11% is only a lower bound on the specificity of TopHat. From: TopHat: discovering splice junctions with RNA-Seq Bioinformatics. 2009;25(9):1105-1111. doi:10.1093/bioinformatics/btp120 Bioinformatics | © 2009 The Author(s)This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

From: TopHat: discovering splice junctions with RNA-Seq Fig. 6. TopHat detects junctions in genes transcribed at very low levels. The gene Pnlip was transcribed at only 7.88 RPKM in the brain tissue according to ERANGE, and yet TopHat reports the complete known gene model. From: TopHat: discovering splice junctions with RNA-Seq Bioinformatics. 2009;25(9):1105-1111. doi:10.1093/bioinformatics/btp120 Bioinformatics | © 2009 The Author(s)This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

From: TopHat: discovering splice junctions with RNA-Seq Fig. 7. A previously unreported splice junction detected by TopHat is shown as the topmost horizontal line. This junction skips two exons in the ADP-ribosylation gene Arfgef1. As explained in Section 2, islands of read coverage in the Bowtie mapping are extended by 45 bp on either side. From: TopHat: discovering splice junctions with RNA-Seq Bioinformatics. 2009;25(9):1105-1111. doi:10.1093/bioinformatics/btp120 Bioinformatics | © 2009 The Author(s)This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.