Hello Lindsey,
Yes, you have this correct. The general path would be to:
- join forward and reverse data per run
- run FASTQ Groomer & FastQC
(note: if your data is already in Sanger FASTQ format with Phred+33
quality scaled
values, the datatype '.fastqsanger' can be directly assigned and the
FASTQ Groomer
step skipped. This is likely true if your data is a from the latest
CASAVA pipeline, but
please double check.)
- discard data as needed based on quality
- split forward and reverse data that passes QC
- concatenate all forward reads from a sample into one FASTQ file
- concatenate all reverse reads from a sample into one FASTQ file.
- for each sample, run TopHat using the two concatenated FASTQ files
To manipulate paired end data, please see the tools -> NGS: QC and
manipulation: FASTQ splitter & FASTQ joiner.
To combined data files head-to-tail from multiple runs into a single
FASTQ file please see the tool -> Text Manipulation: Concatenate datasets.
I am not sure of the actual volume of data, but if these start to get
large or TopHat errors with a memory problem, a local or cluster
instance would be the recommendation: http://getgalaxy.org
For reference:
http://tophat.cbcb.umd.edu/manual.html
http://www.nature.com/nprot/journal/v7/n3/full/nprot.2012.016.html
Hopefully this helps. Others are welcome to post comments/suggestions.
Jen
Galaxy team
On 7/2/12 11:17 AM, Lindsey Kelly wrote:
I am trying to do RNAseq analysis on Paired end data from the
Hiseq2000. I have about 50 files for each sample (25 forward and 25
reverse - although each sample has a different number of files).
I think that I need to:
-convert them into FASTQ sanger format using the FASTSQ groomer tool
-check the quality using the FASTQqc tool
I don't know how to handle this many files. Do I have to groom and run
the QC for each file? Should I join the paired files and run both
tools on each pair, or should I combine all of the data for each
sample (which I don't know how to do) and then groom and run the QC
for all of the reads for the sample.
Thanks in advance for advice
Lindsey
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