gravatar for priscilladraj

2 hours ago by

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.


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