[DOCKTESTERS] BWA-Mem validation of DO51057 (normal BAM only). 96.3% matches, 0.013% miss-matches, and 3.7% soft-matches

Thu Apr 6 10:55:42 EDT 2017

Hi Miguel,

Sounds like a significant achievement! But remind me what a "soft match" is?

Lincoln

On Thu, Apr 6, 2017 at 10:28 AM, Miguel Vazquez <miguel.vazquez at cnio.es>
wrote:

> Dear all,
>
> This is just an advance teaser for the BWA-Mem validation after the latest
> changes, it is currently running over the tumor BAM, but the normal BAM has
> completed and the *missmatches are two orders of magnitude lower* than in
> our two previous attempts. Before further discussion here are the raw
> numbers:
>
> Lines: 1125172217
> Matches: 1083221794
> *Misses: 143716*
> Soft: 41806707
>
> If my calculation are correct this means 96.3% matches, *0.013%
> miss-matches*, and 3.7% soft-matches.
>
> The fix was two part. First realizing that the input of this process
> should not be a single unaligned version of the output BAMs, but several
> input BAMs. Breaking down the output bam into it's constituent BAMs, by a
> process implemented by Jonas, dit not address the problem unfortunately.
> After this first attempt it was pointed out to us, I think by Keiran, that
> the order of the reads matter, and so our attempt to work back from the
> output BAM was not going to work. Junjun came back to us with the second
> part of the fix, he located a subset of original unaligned BAMs in the DKFZ
> that we could use. Downloading these BAM files and submitting them to
> BWA-Mem in the same order as was specified in the output BAM header
> achieved these promising results.
>
> I will reply this message in a few days with the corresponding numbers for
> the other BAM, the tumor, which is currently running.
>
> Best regards
>
> Miguel
>
>
>
> On Sun, Feb 19, 2017 at 1:43 PM, Miguel Vazquez <miguel.vazquez at cnio.es>
> wrote:
>
>> Dear all,
>>
>> Great news! The BWA-Mem test on a real PCAWG donor succeed in running;
>> achieving an overlap with the original BAM alignment similar to the
>> HCC1143 test. The numbers are:
>>
>> Lines: 1708047647
>> Matches: 1589172843
>> Misses: 62726130
>> Soft: 56148674
>>
>> Which mean 93% matches, 3.6% miss-matches, and 3.2% soft-matches.
>> Compared to the HCC1143 test there are a few percentage points in matches
>> that turn into soft-matches (95% and 1.3% to 93% and 3.2%), but the ratio
>> of misses is very close 3.6%.
>>
>> I'm running this test on a second donor.
>>
>> Best regards
>>
>> Miguel
>>
>> On Tue, Feb 14, 2017 at 3:30 PM, Miguel Vazquez <miguel.vazquez at cnio.es>
>> wrote:
>>
>>> 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/bi
>>> n/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>,10:5606682
>>>> 1: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>,10:134948
>>>> 976:N:<DEL>,10:19996638:N:<DEL>
>>>> Missing: 615
>>>>     - Example: 10:101851839:N:<TRA>,10:101851
>>>> 884: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>,1:10906889
>>>> 9: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>,10:104645
>>>> 247:N:<DEL>,10:105097522:N:<DEL>
>>>> Missing: 2908
>>>>     - Example: 10:100107032:N:<TRA>,10:100107
>>>> 151: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>
>>>>
>>>
>>>
>>
>

-- 
*Lincoln Stein*

Scientific Director (Interim), Ontario Institute for Cancer Research
Director, Informatics and Bio-computing Program, OICR
Senior Principal Investigator, OICR
Professor, Department of Molecular Genetics, University of Toronto

<http://goog_1828306398>
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