Hi Laurent:
Using a genome alignment to calculate inner-distance can give misleading
results, due to introns, particularly if you make the calculations directly
from the sam file. It's better to do this with a transcriptome reference
(even a small one, such as rRNA sequences to check contamination). I often
do this with bwa aln/sampe, since the sampe stderr includes lines like:
[infer_isize] inferred external isize from 139003 pairs: 213.886 +/- 65.901
These agree quite well with what you will get from running the Picard tools.
Monica
On Fri, Sep 5, 2014 at 12:21 PM, Laurent MANCHON <[email protected]>
wrote:
> --Hi,
>
> For tophat, i need to fix parameter "-r" i.e. inner distance (instead of
> the insert size) which is insert-length - 2*readLength.
> So, i have mapped my paired-end reads(read length=100) to the reference
> genome with bowtie2, then i used Picard's CollectInsertSizeMetrics.jar
> tool to estimate the insert size
> to then set the mate inner distance parameter in tophat (-r), this is
> the output of picards tools:
>
> MEDIAN_INSERT_SIZE MEDIAN_ABSOLUTE_DEVIATION MIN_INSERT_SIZE
> MAX_INSERT_SIZE MEAN_INSERT_SIZE STANDARD_DEVIATION READ_PAIRS
> PAIR_ORIENTATION W
> IDTH_OF_10_PERCENT WIDTH_OF_20_PERCENT WIDTH_OF_30_PERCENT
> WIDTH_OF_40_PERCENT WIDTH_OF_50_PERCENT WIDTH_OF_60_PERCENT
> WIDTH_OF_70_PERCENT WIDTH_OF_
> 80_PERCENT WIDTH_OF_90_PERCENT WIDTH_OF_99_PERCENT SAMPLE
> LIBRARY READ_GROUP
> 163 38 82 269356442 180.122413 69.468016
> 66668352 FR 15 31 45 61 77 95
> 113 135 247 3777
>
> now if i use another method to compute 'mean insert size' and 'standard
> deviation' directly from the bam file:
> samtools view -F 0x4 File.mapped.bam | awk '{if ($9 >0)
> {sum+=$9;sumsq+=$9*$9;N+=1}} END {print "mean = " sum/N " SD="
> sqrt(sumsq/N - (sum/N)**2)}'
>
> i obtain: mean = 6486.58 SD=806658
>
> In the second method mean insert size is vastly different from that
> obtained in the first method with picard tool.
> Do you think I might be doing something wrong here ? Is there another
> tool to compute the correct mate inner distance ?
>
> thank you --
>
>
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>
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--
Monica Britton
Sr. Bioinformatics Analyst
Genome Center and Bioinformatics Core Facility
University of California, Davis
[email protected]
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