RNA-Seq tophat command error?

Question

[PK]

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

[WouterDeCoster]

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 ;)

[PK]

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.

[Devon Ryan]

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

[PK]

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'!

[Devon Ryan]

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

[PK]

Ok. Thank you very much

Answer 1

[EagleEye]

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