mapping and quantification of paired end reads using BBmap

Question

[rahmati.razieh83]

Hi everyone

I have some paired end RNA -seq reads without reference genome. I assembled them by using trinity. because my reads containing specific Lexogen primers, I have been recommended to use bbmap instead of Tophat2 in order to avoid map rate decrease. I assembled the reads with trinity and at the moment I have trinity.fasta file. For the next step that I need to map reads to the transcriptome and calculate the read count, how can I use bbmap? regarding the existing of lexogen primers in the reads that may reduce the map rate, what is the command of bbmap for mapping paired end reads to trinity.fasta file? and what is the command for calculating reads? Please guide me.

[ahmad mousavi]

if you want to calculate number of mapped reads per transcript the best tools is RSEM which is available in Trinity pipeline

Answer 1

[ahmad mousavi]

Dear Razieh

Edit following script with your own files, try to put all possible adapters and index in adapter.fa file.

I have trimed 5 bases from left and minlen=90 you should change it

for f in *_1.fastq.gz; do  bbmap/bbduk.sh -Xmx20g in1=$f in2=${f/_1./_2.} out1=${f/_1.fastq.gz/_clean_1.fastq.gz} out2=${f/_1.fastq.gz/_clean_2.fastq.gz} ref=adapter.fa ktrim=r hdist=2 k=21 ftl=5  qtrim=20 minlen=90  ; done

You could find more information about bbmap in following page :

http://seqanswers.com/forums/showthread.php?t=42776

[rahmati.razieh83]

Dear ahmad; Thanks alot for your comment. I have already trimmed universal adapters by trimmomatic. But I found out, in addition to adapters my reads contains some random Lexogen primers. because of existing these primers, I have been recommended to use bbmap instead of top hat2 for mapping the reads. Because the reads contains primers and tophat2 will reduce mapping rate, my qustion is that regarding the trinity.fasta file that I have as the assembly output, How can I continue the the process by BBmap? what is the command for mapping the paired end reads to trinity.fasta in a way that I can increase the map rate in comparison with tophat2? I am new in such kind of analysis. please guide me