TopHat, Cufflinks, Cuffdiff

Transcription

1 TopHat, Cufflinks, Cuffdiff Andreas Gisel Institute for Biomedical Technologies - CNR, Bari

2 TopHat

3 TopHat

4 TopHat TopHat is a program that aligns RNA-Seq reads to a genome in order to identify exon-exon splice junctions. It is built on the ultrafast short read mapping program Bowtie. TopHat runs on Linux and OS X

5 TopHat Usage: tophat [options]* <index_base> <reads1_1[,...,readsn_1]> [reads1_2,...readsn_2] Index Input (read1) Input (read2) options: -o/--output-dir <string> Sets the name of the directory in which TopHat will write all of its output. The default is "./tophat_out". -i/--min-intron-length <int> The minimum intron length. TopHat will ignore donor/acceptor pairs closer than this many bases apart. The default is 70. -I/--max-intron-length <int> The maximum intron length. When searching for junctions ab initio, TopHat will ignore donor/acceptor pairs farther than this many bases apart, except when such a pair is supported by a split segment alignment of a long read. The default is p/--num-threads <int> Use this many threads to align reads. The default is 1. --bowtie-n TopHat uses "-v" in Bowtie for initial read mapping (the default), but with this option, "-n" is used instead. Read segments are always mapped using "-v" option. --no-sort-bam Output BAM is not coordinate-sorted.

6 TopHat Usage: tophat [options]* <index_base> <reads1_1[,...,readsn_1]> [reads1_2,...readsn_2] Index Input (read1) Input (read2) example: bowtie-build -f TAIR10_chr1.fas TAIR10_chr1 tophat -o tophat_srr p 2 TAIR10_chr1 options Index SRR505743_rna_AT1G.fastq Input (read1)

7 TopHat Usage: tophat [options]* <index_base> <reads1_1[,...,readsn_1]> [reads1_2,...readsn_2] Index Input (read1) Input (read2) example: bowtie-build -f TAIR10_chr1.fas TAIR10_chr1 tophat -o tophat_srr p 2 TAIR10_chr1 options Index SRR505743_rna_AT1G.fastq Input (read1) results: -rw-rw-r-- 1 embnet embnet Oct 13 10:49 accepted_hits.bam -rw-rw-r-- 1 embnet embnet Oct 13 10:48 deletions.bed -rw-rw-r-- 1 embnet embnet Oct 13 10:49 insertions.bed -rw-rw-r-- 1 embnet embnet Oct 13 10:48 junctions.bed -rw-rw-r-- 1 embnet embnet 72 Oct 13 10:49 left_kept_reads.info drwxrwxr-x 2 embnet embnet 4096 Oct 13 10:49 logs/ -rw-rw-r-- 1 embnet embnet Oct 13 10:48 unmapped_left.fq.z

8 TopHat deletions.bed track name=deletions description="tophat deletions" Chr Chr Chr junctions.bed track name=junctions description="tophat junctions" Chr JUNC ,0,0 2 91,10 0,2569 Chr JUNC ,0,0 2 12,89 0,2490 Chr JUNC ,0,0 2 83,57 0,2561

9 TopHat results: -rw-rw-r-- 1 embnet embnet Oct 13 10:49 accepted_hits.bam -rw-rw-r-- 1 embnet embnet Oct 13 10:48 deletions.bed -rw-rw-r-- 1 embnet embnet Oct 13 10:49 insertions.bed -rw-rw-r-- 1 embnet embnet Oct 13 10:48 junctions.bed -rw-rw-r-- 1 embnet embnet 72 Oct 13 10:49 left_kept_reads.info drwxrwxr-x 2 embnet embnet 4096 Oct 13 10:49 logs/ -rw-rw-r-- 1 embnet embnet Oct 13 10:48 unmapped_left.fq.z deletions.bed track name=deletions description="tophat deletions" Chr Chr Chr junctions.bed track name=junctions description="tophat junctions" Chr JUNC ,0,0 2 91,10 0,2569 Chr JUNC ,0,0 2 12,89 0,2490 Chr JUNC ,0,0 2 83,57 0,2561

10 Cufflinks

11 Cufflinks

12 Cufflinks Usage: cufflinks [options]* <aligned_reads.(sam/bam)> options: -o/--output-dir <string> Sets the name of the directory in which Cufflinks will write all of its output. The default is "./". -p/--num-threads <int> Use this many threads to align reads. The default is 1. -G/--GTF <reference_annotation.(gtf/gff)> Tells Cufflinks to use the supplied reference annotation (a GFF file) to estimate isoform expression. It will not assemble novel transcripts, and the program will ignore alignments not structurally compatible with any reference transcript. example: cufflinks -o cufflinks_srr tophat_srr505743/accepted_hits.bam

13 Cufflinks example: cufflinks -o cufflinks_srr tophat_srr505743/accepted_hits.bam results: -rw-rw-r-- 1 embnet embnet Oct 11 19:24 genes.fpkm_tracking -rw-rw-r-- 1 embnet embnet Oct 11 19:24 isoforms.fpkm_tracking -rw-rw-r-- 1 embnet embnet 0 Oct 11 18:57 skipped.gtf -rw-rw-r-- 1 embnet embnet Oct 11 19:24 transcripts.gtf genes.fpkm_tracking tracking_id class_code nearest_ref_id gene_id gene_short_name tss_id locus length coverage FPKM FPKM_conf_lo FPKM_conf_hi FPKM_status CUFF CUFF Chr1: OK

14 Cuffdiff

15 Cuffdiff

16 Cuffdiff cuffdiff [options]* <transcripts.gtf> <sample1_replicate1.sam[,...,sample1_replicatem]> <sample2_replicate1.sam[,...,sample2_replicatem.sam]>... example: cuffdiff merged_asm/merged.gtf liver1.bam,liver2.bam brain1.bam,brain2.bam cuffdiff annotation.gtf mock_rep1.bam,mock_rep2.bam knockdown_rep1.bam,knockdown_rep2.bam -rw-rw-r-- 1 embnet embnet 53 Oct 11 22:19 bias_params.info -rw-rw-r-- 1 embnet embnet 12 Oct 11 22:19 cds.count_tracking -rw-rw-r-- 1 embnet embnet 115 Oct 11 22:19 cds.diff -rw-rw-r-- 1 embnet embnet 124 Oct 11 22:19 cds_exp.diff -rw-rw-r-- 1 embnet embnet 91 Oct 11 22:19 cds.fpkm_tracking -rw-rw-r-- 1 embnet embnet 115 Oct 11 22:19 cds.read_group_tracking -rw-rw-r-- 1 embnet embnet Oct 11 22:19 gene_exp.diff -rw-rw-r-- 1 embnet embnet Oct 11 22:19 genes.count_tracking -rw-rw-r-- 1 embnet embnet Oct 11 22:19 genes.fpkm_tracking -rw-rw-r-- 1 embnet embnet Oct 11 22:19 genes.read_group_tracking -rw-rw-r-- 1 embnet embnet Oct 11 22:19 isoform_exp.diff -rw-rw-r-- 1 embnet embnet Oct 11 22:19 isoforms.count_tracking -rw-rw-r-- 1 embnet embnet Oct 11 22:19 isoforms.fpkm_tracking -rw-rw-r-- 1 embnet embnet Oct 11 22:19 isoforms.read_group_tracking -rw-rw-r-- 1 embnet embnet Oct 11 22:19 promoters.diff -rw-rw-r-- 1 embnet embnet 228 Oct 11 22:19 read_groups.info -rw-rw-r-- 1 embnet embnet 177 Oct 11 22:19 run.info -rw-rw-r-- 1 embnet embnet Oct 11 22:19 splicing.diff -rw-rw-r-- 1 embnet embnet Oct 11 22:19 tss_group_exp.diff -rw-rw-r-- 1 embnet embnet Oct 11 22:19 tss_groups.count_tracking -rw-rw-r-- 1 embnet embnet Oct 11 22:19 tss_groups.fpkm_tracking -rw-rw-r-- 1 embnet embnet Oct 11 22:19 tss_groups.read_group_tracking -rw-rw-r-- 1 embnet embnet Oct 11 22:07 var_model.info

17 Cuffdiff test_id gene_id gene locus sample_1 sample_2 status value_1 value_2 log2(fold_change) test_stat p_value q_value significant XLOC_ XLOC_ Chr1: q1 q2 OK yes XLOC_ XLOC_ Chr1: q1 q2 OK no