Hello, all
I am a new person in this world and would like your help.
I am trying to analyze Illumina HiSeq2000 results from a nonmodel, rodent both by de novo and genome-based analysis. I am waiting until the de novo results, and meanwhile am trying to map the rodent sequence results to the mouse genome.
I have done the mapping using default settings in Tophat2, but I saw that not much sequences were mapped to the genome, e.g. the accepted_hits.bam were 33.5MB compared to 2.01GB for unmapped.bam for one of my samples.
I have thought possible reasons may be 1) low quality reads getting in the way 2) adapter sequences getting in the way 3) the tophat2 settings were too stringent to compare the two species.
As for 1), when I saw the results from FastQC, I didn't see any problems in the quality of the reads (they were in the green area). As for 2), there were no sequences that were overrepresented, although there were a few k-mers that were enriched in the first 10 bases.
So, I was suspecting that the main culprit was 3) the mapping conditions being too stringent, and I read a few threads here that said that changing a few parameters in bowtie2 may help. I tried mapping the same sequencing results changing the first time, N:0 <- 1, and the second time, N:0 <- 1 & L:20 <- 10, but still had no luck.
So, I'd like to ask, if there are any hints as to what I should change in order to improve mapping/make the threshold more lenient? Specific numbers and variables, published papers with parameters are greatly appreciated!
Thank you in advance!
& Sorry for the long question!
I am a new person in this world and would like your help.
I am trying to analyze Illumina HiSeq2000 results from a nonmodel, rodent both by de novo and genome-based analysis. I am waiting until the de novo results, and meanwhile am trying to map the rodent sequence results to the mouse genome.
I have done the mapping using default settings in Tophat2, but I saw that not much sequences were mapped to the genome, e.g. the accepted_hits.bam were 33.5MB compared to 2.01GB for unmapped.bam for one of my samples.
I have thought possible reasons may be 1) low quality reads getting in the way 2) adapter sequences getting in the way 3) the tophat2 settings were too stringent to compare the two species.
As for 1), when I saw the results from FastQC, I didn't see any problems in the quality of the reads (they were in the green area). As for 2), there were no sequences that were overrepresented, although there were a few k-mers that were enriched in the first 10 bases.
So, I was suspecting that the main culprit was 3) the mapping conditions being too stringent, and I read a few threads here that said that changing a few parameters in bowtie2 may help. I tried mapping the same sequencing results changing the first time, N:0 <- 1, and the second time, N:0 <- 1 & L:20 <- 10, but still had no luck.
So, I'd like to ask, if there are any hints as to what I should change in order to improve mapping/make the threshold more lenient? Specific numbers and variables, published papers with parameters are greatly appreciated!
Thank you in advance!
& Sorry for the long question!
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