Fixed an issue of data that contain tp tag sometimes recognized as flow #8337

ilyasoifer · 2023-05-22T19:38:19Z

droazen · 2023-05-22T21:23:36Z

Thanks @ilyasoifer ! Was the test bam you added aligned with minimap2? If not, we should make sure to add at least one simple HaplotypeCaller regression test that takes an actual minimap2-aligned input.

ilyasoifer · 2023-05-23T06:08:46Z

@droazen the bam that I added was aligned with minimap2. However, it is a really small BAM, so I could not run it through the integration tests. It sounds useful to add minimap2-aligned bam to HaplotypeCallerIntegrationTest. Can you or @jamesemery point me to a suitable BAM for that?

jamesemery · 2023-05-23T17:38:55Z

@ilyasoifer do you have the means to quickly run minimap2? I would recommend simply realigning src/test/resources/large/CEUTrio.HiSeq.WGS.b37.NA12878.20.21.bam with minimap2 and making a quick "are tests consistent with pervious versions" test with a checked in vcf output. I don't know how to wrangle minimap2 to handle mates correctly however so i don't know if this is easy

jamesemery

@ilyasoifer Thank you for doing this. A few more defensive checks and future-proofing to hopefully save us from having this headache again in the future. Could you realign our HaplotypeCaller input bam snippets with minimap 2 and make a quick integration test asserting that they still funciton?

jamesemery · 2023-05-23T17:49:16Z

src/main/java/org/broadinstitute/hellbender/utils/read/FlowBasedReadUtils.java

+            if (readGroup.getPlatform()==null){
+                isFlowPlatform = false;
+            } else {
+                isFlowPlatform = NGSPlatform.valueOf(readGroup.getPlatform()).getSequencerType() == SequencerFlowClass.FLOW;


I know these SequencerFlowClass objects exist and in theory should handle other techonologies, but the codepaths here really only support Ultima. I would change this to check for the Ultima readgroup (so treat potential Iontorrent or 454 data which is also flow) seperately for now so long as they aren't tested. This seems like a recipe for those technologies falling over.

Old Ultima data used PL:LS454 because ULTIMA was not a legal platform. I think I prefer allowing LS454 and ULTIMA to work this way given low chance anyone is now running LS454 data

Can you special case the PL:LS454 platform and give an explanation about it here? Again, I want to be as specific as possible about the inputs here since this is a greedy non opt-in behavior we should be careful we don't start clobbering somebodies inputs in all cases. If it IS labeled as 454 data can you check for the other fields that should make it readable as ultima data?

Added tests

jamesemery · 2023-05-23T17:52:42Z

src/main/java/org/broadinstitute/hellbender/utils/read/FlowBasedReadUtils.java

+                isFlowPlatform = NGSPlatform.valueOf(readGroup.getPlatform()).getSequencerType() == SequencerFlowClass.FLOW;
+            }
+            if (isFlowPlatform) {
+                Utils.nonNull(readGroup);


Given that this is probably the first line of defense for users submitting mis-formatted flow based data to the HaplotypeCaller, can you upgrade these non-null checks to be a little more stringent about expected flow-based header lines and throw exceptions with UserException enforcing the presence of those fields and printing out the readgroup header line in the error message? (and add a quick unit test for the flow platform construction)

I think I fixed this, can you check?

jamesemery · 2023-05-23T17:57:52Z

src/test/java/org/broadinstitute/hellbender/utils/read/FlowBasedReadUtilsUnitTest.java

+        SAMReadGroupRecord rg1 = header.getReadGroup("test");
+        SAMRecord read = reader.iterator().next();
+        GATKRead gread = new SAMRecordToGATKReadAdapter(read);
+        assert(FlowBasedReadUtils.hasFlowTags(gread));


can you record the actual readgroups from that file in a comment here and add a bit of explanation to this test?

jamesemery · 2023-05-23T18:00:33Z

...va/org/broadinstitute/hellbender/tools/walkers/haplotypecaller/AssemblyBasedCallerUtils.java

        final byte minTailQualityToUse = errorCorrectReads ? HaplotypeCallerEngine.MIN_TAIL_QUALITY_WITH_ERROR_CORRECTION : minTailQuality;

        final List<GATKRead> readsToUse = new ArrayList<>();
        final List<GATKRead> hardClippedReadsToUse = new ArrayList<>();

        for (final GATKRead originalRead : region.getReads()) {
+
+            boolean isFlowBasedData = false;
+            if (FlowBasedReadUtils.hasFlowTags(originalRead)) {


Can this be condensed into a single method so external tools don't have to know about the readgroup info caching? Can you make a method FlowBasedReadUtils.isThisFlowBased() that extracts this entire operation away and handles your readgroup caching internally?

ilyasoifer · 2023-05-24T06:18:31Z

@droazen , @jamesemery - I think I addressed your comments, back to you now.
Looking at this deeper, I think we will anyway remove the "removeUncertainFlows" options that triggered all this
It resulted in a need to fix a bug we had in a basecalling, but it had been long resolved.

In any case, I think that the check is more robust now, and it was a good fix.

ilyasoifer · 2023-05-24T06:19:03Z

@ilyasoifer do you have the means to quickly run minimap2? I would recommend simply realigning src/test/resources/large/CEUTrio.HiSeq.WGS.b37.NA12878.20.21.bam with minimap2 and making a quick "are tests consistent with pervious versions" test with a checked in vcf output. I don't know how to wrangle minimap2 to handle mates correctly however so i don't know if this is easy

Added test like this

jamesemery · 2023-05-24T14:24:39Z

src/main/java/org/broadinstitute/hellbender/utils/read/FlowBasedReadUtils.java

+                // we require also FO field to consider ReadGroup to come from the flow
+                boolean tmp = (NGSPlatform.fromReadGroupPL(readGroup.getPlatform()) == NGSPlatform.LS454) ||
+                        (NGSPlatform.fromReadGroupPL(readGroup.getPlatform()) == NGSPlatform.ULTIMA);
+                tmp = tmp & (readGroup!=null);


readGroup!=null here after referencing it several times above?

jamesemery

A few minor comments and one test update and then i think it looks good. Thank you for the quick turnaround on fixing this!

jamesemery · 2023-05-24T14:26:44Z

src/main/java/org/broadinstitute/hellbender/utils/read/FlowBasedReadUtils.java

        return rec.hasAttribute(FlowBasedRead.FLOW_MATRIX_TAG_NAME)
                || rec.hasAttribute(FlowBasedRead.FLOW_MATRiX_OLD_TAG_KR)
                || rec.hasAttribute(FlowBasedRead.FLOW_MATRiX_OLD_TAG_TI);

    }

+    public static boolean isFlowPlatform(final SAMFileHeader hdr, final GATKRead read) {


Can you just add a javadoc to this?

And also possibly update the class javadoc to point people to this method as the definitive "is this flow based and should i trigger that code" method

jamesemery · 2023-05-24T14:27:33Z

.../broadinstitute/hellbender/tools/walkers/haplotypecaller/HaplotypeCallerIntegrationTest.java

+    /*
+     * Test that minimap2 data are supported and consistent with past results
+     */
+    public void testVCFModeMinimap2IsConsistentWithPastResults() throws Exception {


this doesn't get run without adding the @test tag

jamesemery · 2023-05-24T14:32:05Z

src/test/java/org/broadinstitute/hellbender/utils/read/FlowBasedReadUtilsUnitTest.java

+        tests.add(new Object[]{hdr, read3, false});
+
+        GATKRead read4 = makeRead(new byte[]{'T','A','G','C','G','A'}, false);
+        read4.setAttribute("tp",new byte[6]);


This test looks good, can you add in a check for the special cases where Platform=454 +FO tag present and Platform=454 + no FO tag present?

jamesemery · 2023-05-24T14:41:00Z

src/main/java/org/broadinstitute/hellbender/utils/read/FlowBasedReadUtils.java

+                isFlowPlatform = tmp;
+
+                //in addition validate that the ultima data has FO tag, otherwise break
+                if ((NGSPlatform.fromReadGroupPL(readGroup.getPlatform()) == NGSPlatform.ULTIMA) & (!isFlowPlatform)){


the logic here is a little confusing though I believe it accomplishes what you want. You might consider refactoring this a little so its cleaner but its not critical.

jamesemery

A few minor comments and one test update and then i think it looks good. Thank you for the quick turnaround on fixing this!

ilyasoifer · 2023-05-24T19:15:03Z

@jamesemery I believe I addressed your comments, PTAL

jamesemery

One FINAL gripe and then its good to go. Thank you for going back and forth on this.

jamesemery · 2023-05-25T15:06:48Z

src/main/java/org/broadinstitute/hellbender/utils/read/FlowBasedReadUtils.java

+                isFlowPlatform = false;
+            } else if (NGSPlatform.fromReadGroupPL(readGroup.getPlatform())==NGSPlatform.UNKNOWN){
+                isFlowPlatform = false;
+            } else if (NGSPlatform.fromReadGroupPL(readGroup.getPlatform()) == NGSPlatform.LS454) {


I know 454 data is ancient at this point but i would still prefer this LS454 case to explicitly check for

if (readGroup.getFlowOrder()!=null) { isFlowPlatform = true; else false;

I'm not sure i trust the Ultima flow ordering code is going to handle 454 data well without testing it

I think this is not this check is supposed to do. Old Ultima data were labeled LS454 and initially did not have the FO tag. Afterwards the tag was added .
The full check is done by isFlowPlatform function that also verifies that the reads have tp tag.

Ahh i see... horrifying... well i'm alright to let this through in this case then

jamesemery

One FINAL gripe and then its good to go. Thank you for going back and forth on this.

jamesemery

Alright this should be good 👍

ilyasoifer · 2023-05-25T19:43:06Z

Thanks for the help!

AishaShah · 2023-07-12T22:33:11Z

Is this fixed in version 4.4.0.0 of gatk?

droazen · 2023-07-13T16:27:23Z

@AishaShah No, this fix will be part of the next GATK release, which we expect to go out shortly (within the next several weeks).

rmormando · 2023-11-07T18:33:01Z

Hi! I know this issue is closed and a solution has been found but I am facing this same issue right now and wanted to follow up and see if it's been patched in the current 4.4.0.0 version of gatk? Or is the new release expected to be published soon?

ilyasoifer requested review from jamesemery and meganshand May 22, 2023 19:39

jamesemery requested changes May 23, 2023

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ilyasoifer requested review from jamesemery and droazen May 24, 2023 06:16

jamesemery reviewed May 24, 2023

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jamesemery requested changes May 24, 2023

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ilyasoifer requested a review from jamesemery May 24, 2023 19:15

jamesemery reviewed May 25, 2023

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ilyasoifer requested a review from jamesemery May 25, 2023 19:26

ilyasoifer added 8 commits May 25, 2023 22:34

Fixed an issue of data that contain tp tag sometimes recognized as flow

9fb19f3

Added minimap2 data test

096470a

PR comments

06edf28

PR comments

f22faa3

PR comments

a01aa7a

PR comments

9b700d6

Fixed tests

4115227

Fixed tests

5ce230a

ilyasoifer force-pushed the ilyasoifer/fix.tp.requirement.issue-8335 branch from 5395d75 to 5ce230a Compare May 25, 2023 19:35

jamesemery approved these changes May 25, 2023

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ilyasoifer merged commit 4456792 into broadinstitute:master May 26, 2023
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ilyasoifer deleted the ilyasoifer/fix.tp.requirement.issue-8335 branch May 26, 2023 04:34

lh3 mentioned this pull request Nov 2, 2023

Question about SAM output tags lh3/minimap2#25

Closed

ilyasoifer mentioned this pull request Nov 19, 2023

Ultima develop code merge igvteam/igv#1423

Merged

Fixed an issue of data that contain tp tag sometimes recognized as flow #8337

Fixed an issue of data that contain tp tag sometimes recognized as flow #8337

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ilyasoifer commented May 22, 2023

droazen commented May 22, 2023

ilyasoifer commented May 23, 2023

jamesemery commented May 23, 2023

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ilyasoifer commented May 24, 2023

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ilyasoifer commented May 24, 2023

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ilyasoifer commented May 25, 2023

AishaShah commented Jul 12, 2023

droazen commented Jul 13, 2023

rmormando commented Nov 7, 2023