Skip calculation of 50_BP_RULE and add NO_EXON_NUMBER flag if framesh…

[oleraj]

…ift or stop_gain variant doesn't have an exon number listed (usually combined with intronic annotation). #35 and #50

I believe this patch fixes the issue. I tested this and it doesn't report the "uninitialized value in split" error message anymore. I also decided to add a flag "NO_EXON_NUMBER" to alert the user that although this variant includes a 'frameshift' or 'stop_gain' annotation, there is no exon number listed by VEP so this is potentially problematic.

[konradjk]

Looks great! Do you mind adding a short note into the README about what this flag means? After which, happy to merge

[oleraj]

@konradjk let me know if that looks okay.

[sph17]

Hi @oleraj @konradjk, I am running loftee on GRCH38 branch, does this patch also applied to GRCH38? Should I be using the main branch or GRCH38 branch? I am getting the same warning message as issue #35.

Use of uninitialized value in split at /opt/vep/.vep/Plugins/LoF.pm line 562, <__ANONIO__> line 128443. Use of uninitialized value $number_of_exons in subtraction (-) at /opt/vep/.vep/Plugins/LoF.pm line 575, <__ANONIO__> line 128443. Use of uninitialized value in split at /opt/vep/.vep/Plugins/LoF.pm line 562, <__ANONIO__> line 128443. Use of uninitialized value $number_of_exons in subtraction (-) at /opt/vep/.vep/Plugins/LoF.pm line 575, <__ANONIO__> line 128443.

[oleraj]

@sph17 I haven't specifically tested it with GRCh38 but I don't see how it would be different off the top of my head. If you can share an example of a variant that gives this error and what the vep CSQ field looks like for that variant that would be helpful.

[sph17]

Hi @oleraj,

Thanks for getting back to me. Here are the line 128443 if I just run VEP (assuming the line count includes headers)

chr1 27263207 rs138811320 G A 1517.17 PASS AC=1;AF=0.001639;AN=610;AS_BaseQRankSum=-1.2;AS_FS=2.113;AS_FilterStatus=PASS;AS_InbreedingCoeff=0.326;AS_MQ=60;AS_MQRankSum=0;AS_QD=10.68;AS_ReadPosRankSum=0.9;AS_SOR=0.662;AS_VQSLOD=8.7654;AS_culprit=ReadPosRankSum;BaseQRankSum=-1.177;DB;DP=10853;ExcessHet=0;FS=2.113;InbreedingCoeff=0.326;MLEAC=1;MLEAF=0.001639;MQ=60;MQRankSum=0;QD=10.68;ReadPosRankSum=0.955;SOR=0.662;CSQ=A|missense_variant|MODERATE|WDTC1|ENSG00000142784|Transcript|ENST00000319394|protein_coding|3/16||ENST00000319394.8:c.104G>A|ENSP00000317971.3:p.Arg35Gln|523|104|35|R/Q|cGg/cAg|rs138811320|1||1||SNV|1|HGNC|HGNC:29175|YES|NM_001276252.2||1|P4|CCDS60044.1|ENSP00000317971|Q8N5D0.172||UPI000004814E|Q8N5D0-1||tolerated(0.57)|benign(0)|PANTHER:PTHR15574:SF40&PANTHER:PTHR15574|||0.0012|0.0038|0.0014|0|0|0|0.006582|0|0.0003272|0.004696|0.000145|0|0|0|0|0|3.27e-05|0.006582|AA|||||||||,A|missense_variant|MODERATE|WDTC1|ENSG00000142784|Transcript|ENST00000361771|protein_coding|3/16||ENST00000361771.7:c.104G>A|ENSP00000355317.3:p.Arg35Gln|487|104|35|R/Q|cGg/cAg|rs138811320|1||1||SNV|1|HGNC|HGNC:29175||||1|A1|CCDS296.1|ENSP00000355317|Q8N5D0.172||UPI0000070734|Q8N5D0-4||tolerated(0.57)|benign(0)|PANTHER:PTHR15574&PANTHER:PTHR15574:SF40|||0.0012|0.0038|0.0014|0|0|0|0.006582|0|0.0003272|0.004696|0.000145|0|0|0|0|0|3.27e-05|0.006582|AA|||||||||,A|missense_variant&NMD_transcript_variant|MODERATE|WDTC1|ENSG00000142784|Transcript|ENST00000447062|nonsense_mediated_decay|2/16||ENST00000447062.2:c.104G>A|ENSP00000434578.1:p.Arg35Gln|111|104|35|R/Q|cGg/cAg|rs138811320|1||1||SNV|1|HGNC|HGNC:29175||||2|||ENSP00000434578|Q8N5D0.172||UPI00001405BE|Q8N5D0-2||tolerated(0.65)|benign(0.003)||||0.0012|0.0038|0.0014|0|0|0|0.006582|0|0.0003272|0.004696|0.000145|0|0|0|0|0|3.27e-05|0.006582|AA||||||||

I hope this helps.

Thanks, Stephanie

[oleraj]

Hi @sph17 I don't see what about your variant could be causing the issue since the exon number is present for all of the transcripts (i.e., 3/16, 3/16, 2/16). Plus, this variant doesn't have frameshift and intron_variant annotations as the other variants I saw that were causing this error message.
But more importantly, in trying to duplicate this issue, I realized what you mean by GRCH38 branch. I haven't tested this change with the GRCH38 branch of the repo at all and it doesn't appear this commit has been merged with that branch either. It works fine with master, which uses GRCh37. You could try doing liftover of your VCF to GRCh37 and testing with the master branch.

[raonyguimaraes]

I am having the exact same issue here, if I remember correctly converting all the variants to b37 would fix the issue.

@konradjk are you planning on merging changes from master to hg38 branch anytime soon?

time ../../../programs/ensembl-vep/vep -i samplefree.vcf --cache --pick --force_overwrite  --offline --fork 9 --plugin LoF,loftee_path:/home/raony/programs/lofteehg38,human_ancestor_fa:/home/raony/projects/loftee/hg38/human_ancestor.fa.gz,filter_position:0.05,min_intron_size:15,conservation_file:/home/raony/projects/loftee/hg38/loftee.sql,gerp_bigwig:/home/raony/projects/loftee/hg38/gerp_conservation_scores.homo_sapiens.GRCh38.bw --dir_plugins /home/raony/programs/lofteehg38
Smartmatch is experimental at /home/raony/programs/lofteehg38/de_novo_donor.pl line 175.
Smartmatch is experimental at /home/raony/programs/lofteehg38/de_novo_donor.pl line 214.
Smartmatch is experimental at /home/raony/programs/lofteehg38/splice_site_scan.pl line 191.
Smartmatch is experimental at /home/raony/programs/lofteehg38/splice_site_scan.pl line 194.
Smartmatch is experimental at /home/raony/programs/lofteehg38/splice_site_scan.pl line 238.
Smartmatch is experimental at /home/raony/programs/lofteehg38/splice_site_scan.pl line 241.
WARNING: 2101849 : Use of uninitialized value in split at /home/raony/programs/lofteehg38/LoF.pm line 562, <__ANONIO__> line 77808.
Use of uninitialized value $number_of_exons in subtraction (-) at /home/raony/programs/lofteehg38/LoF.pm line 575, <__ANONIO__> line 77808.
WARNING: 2101935 : Use of uninitialized value in split at /home/raony/programs/lofteehg38/LoF.pm line 562, <__ANONIO__> line 87808.
Use of uninitialized value $number_of_exons in subtraction (-) at /home/raony/programs/lofteehg38/LoF.pm line 575, <__ANONIO__> line 87808.
Use of uninitialized value in split at /home/raony/programs/lofteehg38/LoF.pm line 562, <__ANONIO__> line 87808.
Use of uninitialized value $number_of_exons in subtraction (-) at /home/raony/programs/lofteehg38/LoF.pm line 575, <__ANONIO__> line 87808.
WARNING: 2101937 : Use of uninitialized value in split at /home/raony/programs/lofteehg38/LoF.pm line 562, <__ANONIO__> line 87808.
Use of uninitialized value $number_of_exons in subtraction (-) at /home/raony/programs/lofteehg38/LoF.pm line 575, <__ANONIO__> line 87808.
WARNING: 2102345 : Use of uninitialized value in split at /home/raony/programs/lofteehg38/LoF.pm line 562, <__ANONIO__> line 132808.
Use of uninitialized value $number_of_exons in subtraction (-) at /home/raony/programs/lofteehg38/LoF.pm line 575, <__ANONIO__> line 132808.
WARNING: 2102417 : Use of uninitialized value in split at /home/raony/programs/lofteehg38/LoF.pm line 562.
Use of uninitialized value $number_of_exons in subtraction (-) at /home/raony/programs/lofteehg38/LoF.pm line 575.
WARNING: 2106415 : Use of uninitialized value in split at /home/raony/programs/lofteehg38/LoF.pm line 562, <__ANONIO__> line 697808.
Use of uninitialized value $number_of_exons in subtraction (-) at /home/raony/programs/lofteehg38/LoF.pm line 575, <__ANONIO__> line 697808.