Re: [Bio-bwa-help] insert size with bwa

[Dario C. <dco...@em...>]

Tom,

I did reverse complement the reads (from the trimmed fastq file) because 
of the protocol we have. But I did not go through a bam file before 
aligning. My steps were:
- raw fastq
- trim, remove adapters
- reverse complement
- align
- parse pairs

Also, how do you explain the fact that with some tool the sequences 
align and with some don't? And also, what is the difference between a 
flag 81 and 83, for example? Why should I chose only the latter?
Thanks,

Dario



On 05/30/2015 06:18 AM, Thomas W. Blackwell wrote:
>
> An earlier email left one with the suspicion that reverse-strand reads 
> had been reverse-complemented when reverting from .bam to .fastq.  
> This would cause both members in a pair to map to the same strand when 
> re-mapped, so they will not be recognized as "properly paired".  
> Indeed, the numbers shown from flagstat suggested that this was the case.
>
>                                   -  tom blackwell -
>
> On Fri, 29 May 2015, Dario Copetti wrote:
>
>> Hello,
>>
>> I am using bwa to calulate the insert size statistics of a MP library 
>> (40-50 kb insert size). I am having problems analyzing the output 
>> since there are inconsistencies between different commands or tools I 
>> am using.
>>
>> When using bwa mem and samtools flagstat, 97.43% of the sequences 
>> align, of these 83% are properly paired, and the insert size is 143 
>> +-79 bp.
>> When using bwa aln and bwa sampe, only 0.2% of the sequences align 
>> (flags 99 and 83), while the others are 77 (the vast majority), 113, 
>> 65, 97, and 81. At this point, I am not sure of the difference 
>> between codes 81 and 83, for example.
>>
>> Which way would you suggest to go to have the distribution of the 
>> distance between pairs of reads? and also, how come I get such 
>> different results?
>> Thanks,
>>
>> Dario
>>
>>
>>
>>
>> -- 
>> Dario Copetti, PhD
>> Research Associate | Arizona Genomics Institute
>> University of Arizona | BIO5
>>
>> 1657 E. Helen St.
>> Tucson, AZ  85721, USA
>> www.genome.arizona.edu
>>
>>

-- 
Dario Copetti, PhD
Research Associate | Arizona Genomics Institute
University of Arizona | BIO5

1657 E. Helen St.
Tucson, AZ  85721, USA
www.genome.arizona.edu