Entering edit mode
5.3 years ago
skevalkumar
•
0
I am trying to align query sequence with RNA-Seq data (100bp PE). I have tried Bowtie2 and BWA. However, it did not give me reads that are matching or mismatched with query sequence. (I am not interested in aligning RNA-Seq data with Reference genome).
That is fine but why are you not using the
query sequence
as your reference to make an index? Then align your data against it.Note: Doing this always runs into risk of having some reads align in locations that they did not originate from.
I did indexing of my query sequence and tried (--end-to-end and --local parameters) to align with RNA-Seq data. However, overall alignment rate was 0.00%.
What is the length of the query sequence?
length of query sequence -2600bp
I suggest that you try
bbmap.sh
from BBTools (https://sourceforge.net/projects/bbmap/ ). Something like this:This will only write mapped reads to the bam file (this will require
samtools
to be in your path otherwise SAM format will be used). If you only want to see how many reads are aligning then omitout
. All the stats will still be written to STDERR.Was there no alignment even if you used command defaults for
bwa
andbowtie2
?There was no alignment with bowtie2 (all default parameters with endtoend and local) and bwa mem.
Hi, I have tried bbmap.sh and it shows 0.0011% mapped reads. I am reading bbmap reference guide (it is new to me.) thank you for help. Please give me more suggestions.
Not something you want to hear but here goes :
Take 10 reads (from R1 and R2) and blast them at NCBI to confirm that you are looking at the correct sample set and the reads are aligning to right genome. Would eliminate the possibility that you have contamination of some kind.
It is showing almost similar result (0.0008%). I have checked few reads in NCBI. It is matching with my plant species.
The the only plausible explanation is this: