RNA-Seq tophat command error?
Dear friends, I started to learn RNA-seq .I am using this command so please check whether it is right or I need to change anything.
/opt/cufflinks_2_2_0/cufflinks -p 16 -o ~/ACTREC/tophat_cufflinks/sample_1 --library-type fr-firststrand -u -b ~/ACTREC/ref/genome.fa --max-bundle-frags 10000000 --GTF ~/ACTREC/annotation/genes.gtf --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check ~/ACTREC/Tophat/Tophat_P9_Rep1/accepted_hits.bam
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GouthamAtla
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PK •
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PK •
1 answer
Please provide the error you are getting using the command.
Suggestion: Also have a look at this pipeline,
A: Any One please provide protocol for Analysing long noncoding RNA illumina NGS da
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Since you just started learning RNA-seq, let me stop you right here (without checking your command, sorry!). Tophat/Cufflinks is no longer considered the most appropriate toolset for analyzing RNA-seq data. To use a tool somewhat comparable there is the successor pipeline consisting of HISAT2-STRINGTIE-BALLGOWN, see this paper for more information (let me know if you can't get access and I can help.)
An alternative strategy (definitely acceptable) would be to use STAR for alignment (but also HISAT2 works), followed by featureCounts quantification and differential expression analysis using DESeq2, edgeR and/or limma-voom.
Thirdly there are very quick methods using pseudo-mapping such as kallisto and sailfish, which are definitely worth looking into.
PS: it's a "command" not a "comment". That's something else ;)
Thanks for your valuable answer, For my practice, i have to replicate some data. That is why I started from scratch. so please help me.
There's nothing in that command that strikes me as wrong. What error are you getting?
I got reply Loading reference annotation and sequence. No fasta index found for /home/praveenkumarr/ACTREC/ref/genome.fa. Rebuilding, please wait.. Fasta index rebuilt. Warning: couldn't find fasta record for 'chr17_ctg5_hap1'!
So you didn't get an error, just some information printed to the screen. I'm guess that the command completed successfully.
Ok. Thank you very much