I have demultiplexed reads for each of my library. So each libary contains read 1 and read 1 as I used dual indexex. Hence, I combined both reads (read 1 and read 1 specific for each pool) into one fastq.gz file.
As for my reference file, I indexed the fasta file containning all my references, using bowtie2 (I tried using bowtie also previously).
bowtie2-build X.fasta index_X
After that, using bowtie2 I tried align my reads to the sequence and I am not getting any alignments. Below is the command line I used
bowtie1 -x < index file name> -U combined fastq.gz -S aln.sam
I tried using a reference sequence at different lenghth;
- a. 11 mers (partial length)
- b. 89mers (full lenghth)
- c. 30 mers (partial length).
Does reference sequnece lenghth affects my alignment? is my method of alignment correct. My libary should be 89 mers. Since, I could get the full length alignment, I tried to search for two other regions in this 88 mer reference sequence (thats is why I had 11 mer and 30 mer refernce sequennce).
The thing is that, I can see the 11 mer sequence when I search for that sequence in my fasta file. But why bowtie is not looking for it?
And also , when I combine the read 1 and read2 , will bowtie be able to recognose the reverse complement of my reference sequence? Or should align each read with reference sequence at one time ?
Kindly let me know please. I am trying to analyse my first iseq data.