Dear all,
I want to count the reasds from RNA seq by using SALMON tool. I was told that the first thing to do is to create a fasta file with the sequence information (fasta file from reference genome + annotation file.GTF) such as :
(salmon) [@ws7910 RNAseq]$ gffread -w transcripts.fa -g Homo_sapiens.GRCh38.cdna.all.fa Homo_sapiens.GRCh37.75.gtf
I get this result
No fasta index found for Homo_sapiens.GRCh38.cdna.all.fa.
Rebuilding, please wait..
Fasta index rebuilt.
Error creating file: annotation/transcript/transcripts.fa
It creates a file named Homo_sapiens.GRCh38.cdna.all.fa.fai
On the salmon website seems that this step is not necessary so can you please tell me what am I doing wrong? and what is the best way to start using salmon?
I'd prefer to do the counting with BAM files already alignes using TopHat2
Thank you
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Morris_Chair
Hi Morris,
The easiest way to start using salmon is to download the transcriptome file directly (e.g. from Gencode --- ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_29/gencode.v29.transcripts.fa.gz). Then, you can create a salmon index via:
salmon index -t gencode.v29.transcripts.fa.gz -i gencode_v29_idx
and then you can quantify using gencode_v29_idx as the salmon index.
Hi Rob, than you for you answer, why you omit the K-mer size for the index generation? -K
thanks
As a rule of thumb in my experience, the only really crucial thing is that the k-mer length is longer or equal to the read length. k-mer length only has a minor influence on the mapping rate, see Salmon Quantification for RNA-seq Read Pairs with Different Lengths to get a vague idea. Most important factor, given that the library is of high quality without contaminations is the read length which eventually comes down to a proper experimental design. When indexing GENCODE files also mind passing the --gencode option to salmon.
I created the salmon index as you describe and it's ok. I tell you in advance that I'm newe to this and I'm lost for making the code to quantify the reads using gencode_v29_idx.
I have 4 bam files to quantify and I don't know how to formulate the code First, can I run multiple bam files? (two treated and two control for instance) or I'll have to do one by one? I thought that the structure of the code should bee like this but I don't know how to insert the gencode_v29_idx
Salmon quant --libType A -p 4 --validate mapping --seqBias --gcBias -a 1file.bam 2fie.bam 3.file.bam 4fie.bam -o salmon_quant
thank you Rob
probably it's more covenient using fastq filles because there are not others bias
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