Execute a multi-sample 2-pass STAR alignment, sharing the splice junctions across samples. Count fragments per gene and fragments per splice site. Also produce a BAM file with coordinates relative to transcripts. Quantify transcripts in TPM with eXpress. Collate outputs from multiple samples.
snakemake --forceall --dag | dot -Tpng > dag.png
You can run snakemake like this. Notice the --cluster option, used to launch
jobs on an LSF cluster. The Python script bsub.py receives job
scripts from Snakemake and automatically submits them to an appropriate LSF
queue based on job requirements.
snakemake --jobs 999 --cluster '../bsub.py -o stdout'The collated output looks like this:
zcat counts.tsv.gz | head | column -tsample gene_id counts_unstranded counts_strand1 counts_strand2
Sample1 N_unmapped 571 571 571
Sample1 N_multimapping 1384 1384 1384
Sample1 N_noFeature 1178 2059 2087
Sample1 N_ambiguous 141 33 36
Sample1 ENSG00000225630 1 0 1
Sample1 ENSG00000237973 1 1 0
Sample1 ENSG00000248527 1 0 1
Sample1 ENSG00000069424 1 0 1
Sample1 ENSG00000116251 1 1 0
zcat junctions.tsv.gz | head | column -tsample chrom intron_first intron_last strand intron_motif annotated uniquely_mapped_reads multimapped_reads max_spliced_overhang
Sample1 1 6095444 6095529 1 1 1 1 0 19
Sample1 1 6095625 6096635 1 1 1 1 0 30
Sample1 1 10336743 10337073 1 1 1 1 0 33
Sample1 1 16206084 16206179 2 2 1 1 0 24
Sample1 1 16206341 16206947 2 2 1 1 0 8
Sample1 1 23691830 23692608 1 1 1 1 0 22
Sample1 1 23692760 23693806 1 1 1 1 0 31
Sample1 1 23804526 23808134 2 2 1 1 0 12
Sample1 1 27350133 27350388 1 1 1 1 0 28
zcat express.tsv.gz | head | column -tsample bundle_id target_id length eff_length tot_counts uniq_counts est_counts eff_counts ambig_distr_alpha ambig_distr_beta fpkm fpkm_conf_low fpkm_conf_high solvable tpm
Sample1 381 ENST00000216281 3379 3204.675807 1 0 0.500000 0.527198 1.000000e+00 1.000000e+00 7.436703e+01 0.000000e+00 2.231011e+02 F 8.208808e+01
Sample1 381 ENST00000334701 3510 3335.674784 1 0 0.500000 0.526130 1.000000e+00 1.000000e+00 7.144648e+01 0.000000e+00 2.143394e+02 F 7.886431e+01
Sample1 1594 ENST00000215832 11022 10847.616110 2 2 2.000000 2.032152 0.000000e+00 0.000000e+00 8.788003e+01 8.788003e+01 8.788003e+01 T 9.700405e+01
Sample1 1750 ENST00000310144 1579 1404.689867 1 0 0.500000 0.562046 1.000000e+00 1.000000e+00 1.696618e+02 0.000000e+00 5.089854e+02 F 1.872767e+02
Sample1 1750 ENST00000582130 828 653.695733 1 0 0.500000 0.633322 1.000000e+00 1.000000e+00 3.645767e+02 0.000000e+00 1.093730e+03 F 4.024283e+02
Sample1 1762 ENST00000616577 4814 4639.664599 4 0 0.280499 0.291039 1.000000e+00 1.000000e+00 2.881640e+01 0.000000e+00 2.386844e+02 F 3.180822e+01
Sample1 1762 ENST00000530705 4735 4560.665216 4 0 0.310656 0.322531 1.000000e+00 1.000000e+00 3.246727e+01 0.000000e+00 2.562418e+02 F 3.583814e+01
Sample1 1762 ENST00000379056 1101 926.693600 4 0 2.230352 2.649870 1.000000e+00 1.000000e+00 1.147181e+03 0.000000e+00 3.190887e+03 F 1.266285e+03
Sample1 1762 ENST00000528619 731 556.858785 1 0 0.000796 0.001044 1.000000e+00 1.000000e+00 6.809137e-01 0.000000e+00 2.007712e+01 F 7.516087e-01