Hi, 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.
Thanks Pris
You have inserts that are only 89 bp? What is the length of sequencing i.e. reads? What is the size of the reference?
This does not make sense. Are you using two different indexes for the same sample? Why would you do that? You have iSeq data, which would be a small amount of data.
Why do you mean by
combine? Generally the insert sizes are such that the two reads should not overlap in the middle.Aligners automatically reverse complement reads when aligning them so you don't need to do anything.
above it seems you have indexed with bowtie2 but you are running bowtie1 - that wont work