Expression Analysis Pipeline

CoGe can generate gene/transcript expression measurements given a FASTQ input and an annotated genome. Thanks to James Schnable, creator of qTeller, for help developing this pipeline!

When a FASTQ file of sequence reads is loaded in LoadExperiment and associated with an annotated genome, the following analysis steps are performed:

1. The FASTQ file is verified for correct format.
2. CutAdapt is run to trim adapter sequence from the reads (parameters: -q 25 --quality-base=64 -m 17).
3. GMAP is run to index the reference genome sequence.
4. GSNAP is run to align the reads to the reference sequence (parameters: -n 5 --format=sam -Q --gmap-mode=none --nofails).
5. SAMtools is run to compute per-position read depth of the resulting alignment (mpileup -D -Q 20).
6. Cufflinks is run to compte per-transcript FPKM (parameters: -p 24).
7. The per-position read depth and per-transcript FPKM values are log transformed and normalized between 0 and 1 for loading.
8. The three results (raw alignment, per-position read depth, and per-transcript FPKM) are loaded as separate Experiments into a Notebook.

Genomes for which this analysis has been performed can have features imported into qTeller. TBD: how to do this ...

Video Tutorial

• Demo fastq data for Arabidopsis Col-0:
  • 0.17M reads: http://de.iplantcollaborative.org/dl/d/2F807292-34CC-4C8E-96E3-3E668A304D23/test_rna_seq_data_0.17M_reads.fastq
  • 1M reads: http://de.iplantcollaborative.org/dl/d/EFD4F983-80B1-4388-94C4-AD78E73D2795/test_rna_seq_data_1M_reads.fastq
  • 7.6M reads: http://de.iplantcollaborative.org/dl/d/9F6602D6-C66B-4C97-A72A-180AAE55AF95/test_rna_seq_data_7.6M_reads.fastq