[DOCKTESTERS] BWA-Mem validation of HCC1143. 95% matches, 3.6% miss-matches, and 1.3% soft-matches

Tue Feb 14 09:30:43 EST 2017

Dear colleagues,

I'm very happy to say that the BWA-Mem pipeline finished for the HCC1143
data.

I think what solved the problem was setting the headers to the unaligned
BAM files. I'm currently trying it out with the DO35937 donor, but its too
early to say if its working or not.

To compare BAM files I've followed some advice that I found on the internet
https://www.biostars.org/p/166221/. I will detail them a bit below because
I would like some advice as to how appropriate the approach is, but first
here are the numbers:

*Lines*: 74264390
*Matches*: 70565742
*Misses*: 2693687
*Soft*: 1004961

Which means *95% matches, 3.6% miss-matches, and 1.3% soft-matches*.
Matches are when the chromosome and position are the same, soft-matches are
when they are not the same but the position from one of the alignments is
included in the list of alternative positions for the other alignment (e.g
XA:Z:15,-102516528,76M,0), and misses are the rest.

Here is the detailed process from the start. The comparison script is here
https://github.com/mikisvaz/PCAWG-Docker-Test/blob/master/bin/compare_bwa_bam.sh

1) Un-align tumor and normal BAM files, retaining the original aligned BAM
files
2) Run BWA-Mem wich produces a file called HCC1143.merged_output.bam with
alignments from both tumor and normal
3) use samtools to extract the entries, limited for the first in pair (?),
cut the read-name, chromosome, position (??) and extra information (for
additional alignments) and sort them. We do this for the original files and
for the BWA-Mem merged_output file, but separating tumor and normal entries
(marked with the codes 'tumor' and 'normal', I believe from the headers I
set when un-aligning them)
4) join the lines by read-name, separately for the tumor and normal pairs
of files, and check for matches

I've two questions:
(?) Is it OK to select only the first in pair, its what the guy in the
example did, and it did simplify the code without repeated read-names
(??) I guess its OK to only check chromosome and position, the cigar would
be necessarily the same.

Best regards

Miguel

On Mon, Jan 16, 2017 at 3:24 PM, Miguel Vazquez <miguel.vazquez at cnio.es>
wrote:

> Dear all,
>
> Let me summarize the status of the testing for Sanger and DKFZ. The
> validation has been run for two donors for each workflow: DO50311 DO52140
>
> Sanger:
> ----------
>
> Sanger call only somatic variants. The results are *identical for Indels
> and SVs* but *almost identical for SNV.MNV and CNV*. The discrepancies
> are reproducible (on the same machine at least), i.e. the same are found
> after running the workflow a second time.
>
> DKFZ:
> ---------
> DKFZ cals somatic and germline variants, except germline CNVs. For both
> germline and somatic variants the results are *identical for SNV.MNV and
> Indels* but with *large discrepancies for SV and CNV*.
>
> Kortine Kleinheinz and Joachim Weischenfeldt are in the process of
> investigating this issue I believe.
>
> BWA-Mem failed for me and has also failed for Denis Yuen and Jonas
> Demeulemeester. Denis I believe is investigating this problem further. I
> haven't had the chance to investigate this much myself.
>
> Best
>
> Miguel
>
>
>
>
> ---------------------
> RESULTS
> ---------------------
>
> ubuntu at ip-10-253-35-14:~/DockerTest-Miguel$ cat results.txt
>
> Comparison of somatic.snv.mnv for DO50311 using DKFZ
> ---
> Common: 51087
> Extra: 0
> Missing: 0
>
>
> Comparison of somatic.indel for DO50311 using DKFZ
> ---
> Common: 26469
> Extra: 0
> Missing: 0
>
>
> Comparison of somatic.sv for DO50311 using DKFZ
> ---
> Common: 231
> Extra: 44
>     - Example: 10:20596800:N:<TRA>,10:56066821:N:<TRA>,11:16776092:N:<TRA>
> Missing: 48
>     - Example: 10:119704959:N:<INV>,10:131163
> 22:N:<TRA>,10:47063485:N:<TRA>
>
>
> Comparison of somatic.cnv for DO50311 using DKFZ
> ---
> Common: 731
> Extra: 213
>     - Example: 10:132510034:N:<DEL>,10:205968
> 01:N:<NEUTRAL>,10:47674883:N:<NEUTRAL>
> Missing: 190
>     - Example: 10:100891940:N:<NEUTRAL>,10:10
> 4975905:N:<NEUTRAL>,10:119704960:N:<NEUTRAL>
>
>
> Comparison of germline.snv.mnv for DO50311 using DKFZ
> ---
> Common: 3850992
> Extra: 0
> Missing: 0
>
>
> Comparison of germline.indel for DO50311 using DKFZ
> ---
> Common: 709060
> Extra: 0
> Missing: 0
>
>
> Comparison of germline.sv for DO50311 using DKFZ
> ---
> Common: 1393
> Extra: 231
>     - Example: 10:134319313:N:<DEL>,10:134948976:N:<DEL>,10:19996638:N:<
> DEL>
> Missing: 615
>     - Example: 10:101851839:N:<TRA>,10:101851884:N:<TRA>,10:10745225:N:<
> DUP>
>
> File not found /mnt/1TB/work/DockerTest-Migue
> l/tests/DKFZ/DO50311//output//DO50311.germline.cnv.vcf.gz
>
> Comparison of somatic.snv.mnv for DO52140 using DKFZ
> ---
> Common: 37160
> Extra: 0
> Missing: 0
>
>
> Comparison of somatic.indel for DO52140 using DKFZ
> ---
> Common: 19347
> Extra: 0
> Missing: 0
>
>
> Comparison of somatic.sv for DO52140 using DKFZ
> ---
> Common: 72
> Extra: 23
>     - Example: 10:132840774:N:<DEL>,11:382520
> 19:N:<TRA>,11:47700673:N:<TRA>
> Missing: 61
>     - Example: 10:134749140:N:<DEL>,11:179191:N:<TRA>,11:38252005:N:<TRA>
>
>
> Comparison of somatic.cnv for DO52140 using DKFZ
> ---
> Common: 275
> Extra: 94
>     - Example: 1:106505931:N:<LOH>,1:109068899:N:<DEL>,1:109359995:N:<DEL>
> Missing: 286
>     - Example: 10:88653561:N:<LOH>,11:179192:N:<LOH>,11:38252006:N:<LOH>
>
>
> Comparison of germline.snv.mnv for DO52140 using DKFZ
> ---
> Common: 3833896
> Extra: 0
> Missing: 0
>
>
> Comparison of germline.indel for DO52140 using DKFZ
> ---
> Common: 706572
> Extra: 0
> Missing: 0
>
>
> Comparison of germline.sv for DO52140 using DKFZ
> ---
> Common: 1108
> Extra: 1116
>     - Example: 10:102158308:N:<DEL>,10:104645247:N:<DEL>,10:105097522:N:<
> DEL>
> Missing: 2908
>     - Example: 10:100107032:N:<TRA>,10:100107151:N:<TRA>,10:102158345:N:<
> DEL>
>
> File not found /mnt/1TB/work/DockerTest-Migue
> l/tests/DKFZ/DO52140//output//DO52140.germline.cnv.vcf.gz
>
> Comparison of somatic.snv.mnv for DO50311 using Sanger
> ---
> Common: 156299
> Extra: 1
>     - Example: Y:58885197:A:G
> Missing: 14
>     - Example: 1:102887902:A:T,1:143165228:C:G,16:87047601:A:C
>
>
> Comparison of somatic.indel for DO50311 using Sanger
> ---
> Common: 812487
> Extra: 0
> Missing: 0
>
>
> Comparison of somatic.sv for DO50311 using Sanger
> ---
> Common: 260
> Extra: 0
> Missing: 0
>
>
> Comparison of somatic.cnv for DO50311 using Sanger
> ---
> Common: 138
> Extra: 0
> Missing: 0
>
>
> Comparison of somatic.snv.mnv for DO52140 using Sanger
> ---
> Common: 87234
> Extra: 5
>     - Example: 1:23719098:A:G,12:43715930:T:A,20:4058335:T:A
> Missing: 7
>     - Example: 10:6881937:A:T,1:148579866:A:G,11:9271589:T:A
>
>
> Comparison of somatic.indel for DO52140 using Sanger
> ---
> Common: 803986
> Extra: 0
> Missing: 0
>
>
> Comparison of somatic.sv for DO52140 using Sanger
> ---
> Common: 6
> Extra: 0
> Missing: 0
>
>
> Comparison of somatic.cnv for DO52140 using Sanger
> ---
> Common: 36
> Extra: 0
> Missing: 2
>     - Example: 10:11767915:T:<CNV>,10:11779907:G:<CNV>
>
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