Annotation of double base substitutions (DBS,DNP)

[andreyurch]

I found that DBS are not annotated correctly. For example:

12 115109872 115109873 GG AA (Hg19)

annotated as "nonframeshift substitution" while the main outcome is nonsynonymous mutation in TBX3 gene. Moreover, any other important annotations of mutational effect like Polyphen, CADD, GERP and others are not provided for DBS.

[kaichop]

what is the command line that you used?

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On Fri, Feb 23, 2024 at 6:56 AM Andrey Yurchenko ***@***.***> wrote: I found that DBS are not annotated correctly. For example: 12 115109872 115109873 GG AA (Hg19) annotated as "nonframeshift substitution" while the main outcome is nonsynonymous mutation in TBX3 gene. Moreover, any other important annotations of mutational effect like Polyphen, CADD, GERP and others are not provided for DBS. — Reply to this email directly, view it on GitHub <#239>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ABNG3OE6BH2G3ADX2D36HZDYVB7W7AVCNFSM6AAAAABDWS5QJCVHI2DSMVQWIX3LMV43ASLTON2WKOZSGE2TAOJTHA2DMNQ> . You are receiving this because you are subscribed to this thread.Message ID: ***@***.***>

[andreyurch]

table_annovar.pl test.txt /humandb -buildver hg19 -out test_out.txt -remove -protocol refGene,dbnsfp35a -operation g,f -nastring . --thread 1 --maxgenethread 1

[kaichop]

I see. Since the mutation is not a single nucleotide mutation, it cannot be called as a nonsynonymous mutation which is defined only for SNPs in ANNOVAR. For substitutions, they are classified as nonframeshift or frameshift. There is no automated workaround for this issue, to change table_annovar output. However, if you remove the "-remove" argument in your command, then you can use something below to get the protein changes as well as amino acid changes. coding_change.pl test_out.txt.refGene.exonic_variant_function.orig humandb/hg19_refGene.txt humandb/hg19_refGeneMrna.fa

line1 NM_005996 WILDTYPE

MSLSMRDPVIPGTSMAYHPFLPHRAPDFAMSAVLGHQPPFFPALTLPPNGAAALSLPGALAKPIMDQLVGAAETGIPFSSLGPQAHLRPLKTMEPEEEVE DDPKVHLEAKELWDQFHKRGTEMVITKSGRRMFPPFKVRCSGLDKKAKYILLMDIIAADDCRYKFHNSRWMVAGKADPEMPKRMYIHPDSPATGEQWMSK VVTFHKLKLTNNISDKHGFTILNSMHKYQPRFHIVRANDILKLPYSTFRTYLFPETEFIAVTAYQNDKITQLKIDNNPFAKGFRDTGNGRREKRKQLTLQ SMRVFDERHKKENGTSDESSSEQAAFNCFAQASSPAASTVGTSNLKDLCPSEGESDAEAESKEEHGPEACDAAKISTTTSEEPCRDKGSPAVKAHLFAAE RPRDSGRLDKASPDSRHSPATISSSTRGLGAEERRSPVREGTAPAKVEEARALPGKEAFAPLTVQTDAAAAHLAQGPLPGLGFAPGLAGQQFFNGHPLFL HPSQFAMGGAFSSMAAAGMGPLLATVSGASTGVSGLDSTAMASAAAAQGLSGASAATLPFHLQQHVLASQGLAMSPFGSLFPYPYTYMAAAAAASSAAAS SSVHRHPFLNLNTMRPRLRYSPYSIPVPVPDGSSLLTTALPSMAAAAGPLDGKVAALAASPASVAVDSGSELNSRSSTLSSSSMSLSPKLCAEKEAATSE LQSIQRLVSGLEAKPDRSRSASP*

line1 NM_005996 c.1945_1946delinsTT p.P649F protein-altering (position

649 changed from P to F) MSLSMRDPVIPGTSMAYHPFLPHRAPDFAMSAVLGHQPPFFPALTLPPNGAAALSLPGALAKPIMDQLVGAAETGIPFSSLGPQAHLRPLKTMEPEEEVE DDPKVHLEAKELWDQFHKRGTEMVITKSGRRMFPPFKVRCSGLDKKAKYILLMDIIAADDCRYKFHNSRWMVAGKADPEMPKRMYIHPDSPATGEQWMSK VVTFHKLKLTNNISDKHGFTILNSMHKYQPRFHIVRANDILKLPYSTFRTYLFPETEFIAVTAYQNDKITQLKIDNNPFAKGFRDTGNGRREKRKQLTLQ SMRVFDERHKKENGTSDESSSEQAAFNCFAQASSPAASTVGTSNLKDLCPSEGESDAEAESKEEHGPEACDAAKISTTTSEEPCRDKGSPAVKAHLFAAE RPRDSGRLDKASPDSRHSPATISSSTRGLGAEERRSPVREGTAPAKVEEARALPGKEAFAPLTVQTDAAAAHLAQGPLPGLGFAPGLAGQQFFNGHPLFL HPSQFAMGGAFSSMAAAGMGPLLATVSGASTGVSGLDSTAMASAAAAQGLSGASAATLPFHLQQHVLASQGLAMSPFGSLFPYPYTYMAAAAAASSAAAS SSVHRHPFLNLNTMRPRLRYSPYSIPVPVPDGSSLLTTALPSMAAAAGFLDGKVAALAASPASVAVDSGSELNSRSSTLSSSSMSLSPKLCAEKEAATSE LQSIQRLVSGLEAKPDRSRSASP*

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On Fri, Feb 23, 2024 at 9:07 AM Andrey Yurchenko ***@***.***> wrote: table_annovar.pl test.txt /humandb -buildver hg19 -out test_out.txt -remove -protocol refGene,dbnsfp35a -operation g,f -nastring . --thread 1 --maxgenethread 1 — Reply to this email directly, view it on GitHub <#239 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ABNG3OFUOX7EYPSQH5VK3NLYVCPBNAVCNFSM6AAAAABDWS5QJCVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMYTSNRRGM4DSMBTGU> . You are receiving this because you commented.Message ID: ***@***.***>

[andreyurch]

Thank you for the answer. I believe that it is a serious issue because in cancers we often see double base substitutions and they need to be annotated accordingly if they change amino acid.

[kaichop]

I just run your command. As I said it cannot be called as a nonsynonymous mutation due to not being SNP. But if your concern is only whether an amino acid is changed, then you can check the AAChange column in annovar output. ***@***.*** ]$ cat test_out.txt.hg19_multianno.txt Chr Start End Ref Alt Func.refGene Gene.refGene GeneDetail.refGene ExonicFunc.refGene AAChange.refGene 12 115109872 115109873 GG AA exonic TBX3 . nonframeshift substitution TBX3:NM_005996:exon7:c.1945_1946delinsTT:p.P649F,TBX3:NM_016569:exon8:c.2005_2006delinsTT:p.P669F

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On Fri, Feb 23, 2024 at 11:08 AM Andrey Yurchenko ***@***.***> wrote: Thank you for the answer. I believe that it is a serious issue because in cancers we often see double base substitutions and they need to be annotated accordingly if they change amino acid. — Reply to this email directly, view it on GitHub <#239 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ABNG3OH6PA4XFURQDCNCJIDYVC5GFAVCNFSM6AAAAABDWS5QJCVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMYTSNRRGU4TSNRVGU> . You are receiving this because you commented.Message ID: ***@***.***>