Question: RNA-Seq tophat command error?
0
gravatar for barrypraveen
2.6 years ago by
barrypraveen20
barrypraveen20 wrote:

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

ADD COMMENTlink modified 2.6 years ago by geek_y9.8k • written 2.6 years ago by barrypraveen20
1

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 ;)

ADD REPLYlink modified 2.6 years ago • written 2.6 years ago by WouterDeCoster40k

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.

ADD REPLYlink written 2.6 years ago by barrypraveen20
1

There's nothing in that command that strikes me as wrong. What error are you getting?

ADD REPLYlink written 2.6 years ago by Devon Ryan91k

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'!

ADD REPLYlink modified 2.6 years ago • written 2.6 years ago by barrypraveen20
1

So you didn't get an error, just some information printed to the screen. I'm guess that the command completed successfully.

ADD REPLYlink written 2.6 years ago by Devon Ryan91k

Ok. Thank you very much

ADD REPLYlink written 2.6 years ago by barrypraveen20
4
gravatar for EagleEye
2.6 years ago by
EagleEye6.4k
Sweden
EagleEye6.4k wrote:

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

ADD COMMENTlink modified 2.6 years ago • written 2.6 years ago by EagleEye6.4k
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