cDNA level annotation

Annotation from cDNA level is handled by the canno subcommand.

cDNA region

$ transvar canno --ccds -i 'ABCB11:c.1198-8_1202'

outputs

ABCB11:c.1198-8_1202 CCDS46444 (protein_coding)      ABCB11  -
   chr2:g.169833193_169833205GGTTTCTGGAGTG/c.1198-8_1202CACTCCAGAAACC/p.400_401KP    from_[cds_in_exon_11]_to_[intron_between_exon_10_and_11]
   C2=acceptor_splice_site_on_exon_11_at_chr2:169833198_included;source=CCDS

cDNA variant

Single Nucleotide Variation (SNV)

TransVar infers nucleotide mutation through PIK3CA:c.1633G>A. Note that nucleotide identity follows the natural sequence, i.e., if transcript is interpreted on the reverse-complementary strand, the base at the site needs to be reverse-complemented too.

$ transvar canno --ccds -i 'PIK3CA:c.1633G>A'

outputs

PIK3CA:c.1633G>A     CCDS43171 (protein_coding)      PIK3CA  +
   chr3:g.178936091G>A/c.1633G>A/p.E545K     inside_[cds_in_exon_9]
   CSQN=Missense;dbsnp=rs104886003(chr3:178936091G>A);reference_codon=GAG;altern
   ative_codon=AAG;source=CCDS

The SNV can be in the intronic region, e.g.,

$ transvar canno --ccds -i 'ABCB11:c.1198-8C>A'

outputs

ABCB11:c.1198-8C>A   CCDS46444 (protein_coding)      ABCB11  -
   chr2:g.169833205G>T/c.1198-8C>A/. inside_[intron_between_exon_10_and_11]
   CSQN=IntronicSNV;source=CCDS

Or in the 5’-UTR region, e.g.,

$ transvar canno -i 'KCNJ11:c.-134G>T' --ensembl
KCNJ11:c.-134G>T     ENST00000339994 (protein_coding)        KCNJ11  -
   chr11:g.17409772C>A/c.1-134G>T/.  inside_[5-UTR;noncoding_exon_1]
   CSQN=5-UTRSNV;dbsnp=rs387906398(chr11:17409772C>A);aliases=ENSP00000345708;so
   urce=Ensembl

Or in the 3’-UTR region, e.g.,

$ transvar canno -i 'MSH2:c.*95C>T' --refseq
MSH2:c.*95C>T        NM_000251 (protein_coding)      MSH2    +
   chr2:g.47710183C>T/c.*95C>T/.     inside_[3-UTR;noncoding_exon_16]
   CSQN=3-UTRSNV;dbsnp=rs587779062(chr2:47710183C>T);dbxref=GeneID:4436,HGNC:732
   5,HPRD:00389,MIM:609309;aliases=NP_000242;source=RefSeq
MSH2:c.*95C>T        NM_001258281 (protein_coding)   MSH2    +
   chr2:g.47710183C>T/c.*95C>T/.     inside_[3-UTR;noncoding_exon_17]
   CSQN=3-UTRSNV;dbsnp=rs587779062(chr2:47710183C>T);dbxref=GeneID:4436,HGNC:732
   5,HPRD:00389,MIM:609309;aliases=NP_001245210;source=RefSeq
MSH2:c.*95C>T        XM_005264333 (protein_coding)   MSH2    +
   chr2:g.47710183C>T/c.*95C>T/.     inside_[3-UTR;noncoding_exon_15]
   CSQN=3-UTRSNV;dbsnp=rs587779062(chr2:47710183C>T);dbxref=GeneID:4436,HGNC:732
   5,HPRD:00389,MIM:609309;aliases=XP_005264390;source=RefSeq

insertion

An insertion may result in: 1) a pure insertion of amino acids; 2) a block substitution of amino acids, when insertion occur after 1st or 2nd base in a codon; or 3) a frame-shift. Following HGVS nomenclature, TransVar labels the first different amino acid and the length of the peptide util stop codon, assuming no change in the splicing.

Example: to annotate an in-frame, in-phase insertion,

$ transvar canno --ccds -i 'ACIN1:c.1932_1933insATTCAC'
ACIN1:c.1932_1933insATTCAC   CCDS9587 (protein_coding)       ACIN1   -
   chr14:g.23548785_23548786insGTGAAT/c.1932_1933insATTCAC/p.R644_S645insIH  inside_[cds_in_exon_6]
   CSQN=InFrameInsertion;left_align_gDNA=g.23548785_23548786insGTGAAT;unalign_gD
   NA=g.23548785_23548786insGTGAAT;left_align_cDNA=c.1932_1933insATTCAC;unalign_
   cDNA=c.1932_1933insATTCAC;left_align_protein=p.R644_S645insIH;unalign_protein
   =p.R644_S645insIH;phase=0;source=CCDS
ACIN1:c.1932_1933insATTCAC   CCDS53889 (protein_coding)      ACIN1   -
   chr14:g.23548157_23548158insGTGAAT/c.1932_1933insATTCAC/p.P644_V645insIH  inside_[cds_in_exon_6]
   CSQN=InFrameInsertion;left_align_gDNA=g.23548157_23548158insGTGAAT;unalign_gD
   NA=g.23548157_23548158insGTGAAT;left_align_cDNA=c.1932_1933insATTCAC;unalign_
   cDNA=c.1932_1933insATTCAC;left_align_protein=p.P644_V645insIH;unalign_protein
   =p.P644_V645insIH;phase=0;source=CCDS
ACIN1:c.1932_1933insATTCAC   CCDS55905 (protein_coding)      ACIN1   -
   chr14:g.23548785_23548786insGTGAAT/c.1932_1933insATTCAC/p.R644_S645insIH  inside_[cds_in_exon_6]
   CSQN=InFrameInsertion;left_align_gDNA=g.23548785_23548786insGTGAAT;unalign_gD
   NA=g.23548785_23548786insGTGAAT;left_align_cDNA=c.1932_1933insATTCAC;unalign_
   cDNA=c.1932_1933insATTCAC;left_align_protein=p.R644_S645insIH;unalign_protein
   =p.R644_S645insIH;phase=0;source=CCDS

Phase = 0,1,2 indicates whether the insertion happen after the 3rd, 1st or 2nd base of a codon, respectively. An insertion in phase refers to one with Phase=0.

Example: to annotate an out-of-phase, in-frame insertion,

$ transvar canno --ccds -i 'ACIN1:c.1930_1931insATTCAC'
ACIN1:c.1930_1931insATTCAC   CCDS9587 (protein_coding)       ACIN1   -
   chr14:g.23548792_23548793insTGTGAA/c.1930_1931insATTCAC/p.S643_R644insHS  inside_[cds_in_exon_6]
   CSQN=InFrameInsertion;left_align_gDNA=g.23548787_23548788insGTGAAT;unalign_gD
   NA=g.23548787_23548788insGTGAAT;left_align_cDNA=c.1925_1926insTTCACA;unalign_
   cDNA=c.1930_1931insATTCAC;left_align_protein=p.R642_S643insSH;unalign_protein
   =p.S643_R644insHS;phase=1;source=CCDS
ACIN1:c.1930_1931insATTCAC   CCDS53889 (protein_coding)      ACIN1   -
   chr14:g.23548162_23548163insAATGTG/c.1930_1931insATTCAC/p.P643_P644insHS  inside_[cds_in_exon_6]
   CSQN=InFrameInsertion;left_align_gDNA=g.23548159_23548160insGTGAAT;unalign_gD
   NA=g.23548159_23548160insGTGAAT;left_align_cDNA=c.1927_1928insCACATT;unalign_
   cDNA=c.1930_1931insATTCAC;left_align_protein=p.P643_P644insHS;unalign_protein
   =p.P643_P644insHS;phase=1;source=CCDS
ACIN1:c.1930_1931insATTCAC   CCDS55905 (protein_coding)      ACIN1   -
   chr14:g.23548792_23548793insTGTGAA/c.1930_1931insATTCAC/p.S643_R644insHS  inside_[cds_in_exon_6]
   CSQN=InFrameInsertion;left_align_gDNA=g.23548787_23548788insGTGAAT;unalign_gD
   NA=g.23548787_23548788insGTGAAT;left_align_cDNA=c.1925_1926insTTCACA;unalign_
   cDNA=c.1930_1931insATTCAC;left_align_protein=p.R642_S643insSH;unalign_protein
   =p.S643_R644insHS;phase=1;source=CCDS

Reverse annotation can result in different identifiers after left/right alignments, e.g.,

$ transvar canno --ccds -i 'AATK:c.3976_3977insCGCCCA'

results in

AATK:c.3976_3977insCGCCCA    CCDS45807 (protein_coding)      AATK    -
   chr17:g.79093282_79093287dupTGGGCG/c.3988_3993dupACGCCC/p.T1330_P1331dupTP        inside_[cds_in_exon_13]
   CSQN=InFrameInsertion;left_align_gDNA=g.79093270_79093271insGGGCGT;unalign_gD
   NA=g.79093282_79093287dupTGGGCG;left_align_cDNA=c.3976_3977insCGCCCA;unalign_
   cDNA=c.3976_3977insCGCCCA;left_align_protein=p.A1326_P1327insPT;unalign_prote
   in=p.A1326_P1327insPT;phase=1;source=CCDS

Note how insertion switch to duplication when 5’flanking is identical. This conforms to HGVS recommendation to replace insertion notation with duplication when possible.

Example: to annotate a frame-shift insertion, frameshift mutations have not alternative alignments. Hence only cDNA and gDNA have left alignment and unalignment reports.

$ transvar canno --ccds -i 'AAAS:c.1225_1226insG'

results in

AAAS:c.1225_1226insG CCDS8856 (protein_coding)       AAAS    -
   chr12:g.53702093dupC/c.1225dupG/p.E409Gfs*17      inside_[cds_in_exon_13]
   CSQN=Frameshift;left_align_gDNA=g.53702089_53702090insC;unalign_gDNA=g.537020
   89_53702090insC;left_align_cDNA=c.1221_1222insG;unalign_cDNA=c.1225dupG;sourc
   e=CCDS
AAAS:c.1225_1226insG CCDS53797 (protein_coding)      AAAS    -
   chr12:g.53701842_53701843insC/c.1225_1226insG/p.L409Rfs*54        inside_[cds_in_exon_13]
   CSQN=Frameshift;left_align_gDNA=g.53701842_53701843insC;unalign_gDNA=g.537018
   42_53701843insC;left_align_cDNA=c.1225_1226insG;unalign_cDNA=c.1225_1226insG;
   source=CCDS

Example: to annotate an intronic insertion,

$ transvar canno --ccds -i 'ADAM33:c.991-3_991-2insC'

outputs

ADAM33:c.991-3_991-2insC     CCDS13058 (protein_coding)      ADAM33  -
   chr20:g.3654151dupG/c.991-3dupC/. inside_[intron_between_exon_10_and_11]
   CSQN=IntronicInsertion;left_align_gDNA=g.3654145_3654146insG;unalign_gDNA=g.3
   654145_3654146insG;left_align_cDNA=c.991-9_991-8insC;unalign_cDNA=c.991-3dupC
   ;source=CCDS

In the case of intronic insertions, amino acid identifier is not applicable, represented in a .. But cDNA and gDNA identifier are right-aligned according to their natural order, respecting HGVS nomenclature.

Insertion could occur to splice sites. TransVar identifies such cases and report splice site and repress translation of protein change.

$ transvar canno --ccds -i 'ADAM33:c.991_992insC'

results in

ADAM33:c.991_992insC CCDS13058 (protein_coding)      ADAM33  -
   chr20:g.3654142_3654143insG/c.991_992insC/.       inside_[cds_in_exon_11]
   CSQN=SpliceAcceptorInsertion;left_align_gDNA=g.3654142_3654143insG;unalign_gD
   NA=g.3654142_3654143insG;left_align_cDNA=c.991_992insC;unalign_cDNA=c.991_992
   insC;C2=acceptor_splice_site_on_exon_11_at_chr20:3654144_affected;source=CCDS

deletion

Similar to insertions, deletion can be in-frame or frame-shift. The consequence of deletion to amino acid sequence may appear a simple deletion or a block substitution (in the case where in-frame deletion is out of phase, i.e., partially delete codons).

Example: to annotate an in-frame deletion,

$ transvar canno --ccds -i 'A4GNT:c.694_696delTTG'
A4GNT:c.694_696delTTG        CCDS3097 (protein_coding)       A4GNT   -
   chr3:g.137843435_137843437delACA/c.694_696delTTG/p.L232delL       inside_[cds_in_exon_2]
   CSQN=InFrameDeletion;left_align_gDNA=g.137843433_137843435delCAA;unaligned_gD
   NA=g.137843433_137843435delCAA;left_align_cDNA=c.692_694delTGT;unalign_cDNA=c
   .694_696delTTG;left_align_protein=p.L232delL;unalign_protein=p.L232delL;sourc
   e=CCDS

Example: to annotate a in-frame, out-of-phase deletion,

$ transvar canno --ccds -i 'ABHD15:c.431_433delGTG'
ABHD15:c.431_433delGTG       CCDS32602 (protein_coding)      ABHD15  -
   chr17:g.27893552_27893554delCAC/c.431_433delGTG/p.C144_V145delinsF        inside_[cds_in_exon_1]
   CSQN=MultiAAMissense;left_align_gDNA=g.27893552_27893554delCAC;unaligned_gDNA
   =g.27893552_27893554delCAC;left_align_cDNA=c.431_433delGTG;unalign_cDNA=c.431
   _433delGTG;source=CCDS

Example: to annotate a frame-shift deletion,

$ transvar canno --ccds -i 'AADACL3:c.374delG'
AADACL3:c.374delG    CCDS41252 (protein_coding)      AADACL3 +
   chr1:g.12785494delG/c.374delG/p.C125Ffs*17        inside_[cds_in_exon_3]
   CSQN=Frameshift;left_align_gDNA=g.12785494delG;unaligned_gDNA=g.12785494delG;
   left_align_cDNA=c.374delG;unalign_cDNA=c.374delG;source=CCDS

Example: to annotate a deletion that span from intronic to coding region, protein prediction is suppressed due to loss of splice site.

$ transvar canno --ccds -i 'ABCB11:c.1198-8_1199delcactccagAA'
ABCB11:c.1198-8_1199delcactccagAA    CCDS46444 (protein_coding)      ABCB11  -
   chr2:g.169833196_169833205delTTCTGGAGTG/c.1198-8_1199delCACTCCAGAA/.      from_[cds_in_exon_11]_to_[intron_between_exon_10_and_11]
   CSQN=SpliceAcceptorDeletion;left_align_gDNA=g.169833196_169833205delTTCTGGAGT
   G;unaligned_gDNA=g.169833196_169833205delTTCTGGAGTG;left_align_cDNA=c.1198-8_
   1199delCACTCCAGAA;unalign_cDNA=c.1198-8_1199delCACTCCAGAA;C2=acceptor_splice_
   site_on_exon_11_at_chr2:169833198_lost;source=CCDS

block substitution

Example: to annotate a block substitution in coding region,

$ transvar canno --ccds -i 'A1CF:c.508_509delinsTT'
A1CF:c.508_509delinsTT       CCDS7241 (protein_coding)       A1CF    -
   chr10:g.52595929_52595930delinsAA/c.508_509delinsTT/p.P170L       inside_[cds_in_exon_4]
   CSQN=Missense;codon_cDNA=508-509-510;source=CCDS
A1CF:c.508_509delinsTT       CCDS7242 (protein_coding)       A1CF    -
   chr10:g.52595929_52595930delinsAA/c.508_509delinsTT/p.P170L       inside_[cds_in_exon_4]
   CSQN=Missense;codon_cDNA=508-509-510;source=CCDS
A1CF:c.508_509delinsTT       CCDS7243 (protein_coding)       A1CF    -
   chr10:g.52595953_52595954delinsAA/c.508_509delinsTT/p.G170F       inside_[cds_in_exon_4]
   CSQN=Missense;codon_cDNA=508-509-510;source=CCDS

Block substitution does not necessarily results in block substitution in amino acid. For example, the following substitution results in a deletion, where protein alternative alignment should be reported.

$ transvar canno --ccds -i 'CSRNP1:c.1212_1224delinsGGAGGAGGAA'
CSRNP1:c.1212_1224delinsGGAGGAGGAA   CCDS2682 (protein_coding)       CSRNP1  -
   chr3:g.39185092_39185104delinsTTCCTCCTCC/c.1212_1224delinsGGAGGAGGAA/p.E411delE   inside_[cds_in_exon_4]
   CSQN=InFrameDeletion;begin_codon_cDNA=1210-1211-1212;end_codon_cDNA=1222-1223
   -1224;left_align_protein=p.E405delE;unalign_protein=p.E408delE;source=CCDS

Likewise, block substitution could occur to intronic region,

$ transvar canno --ccds -i 'A1CF:c.1460+2_1460+3delinsCC'
A1CF:c.1460+2_1460+3delinsCC CCDS7241 (protein_coding)       A1CF    -
   chr10:g.52570797_52570798delinsGG/c.1460+2_1460+3delinsCC/.       inside_[intron_between_exon_9_and_10]
   CSQN=IntronicBlockSubstitution;source=CCDS

When block substitution occurs across splice site, TransVar put a tag in the info fields and does not predict amino acid change.

$ transvar canno --ccds -i 'A1CF:c.1459_1460+3delinsCC'
A1CF:c.1459_1460+3delinsCC   CCDS7241 (protein_coding)       A1CF    -
   chr10:g.52570797_52570801delinsGG/c.1459_1460+3delinsCC/. from_[intron_between_exon_9_and_10]_to_[cds_in_exon_9]
   CSQN=SpliceDonorBlockSubstitution;C2=donor_splice_site_on_exon_9_at_chr10:525
   70799_lost;source=CCDS

duplication

Duplication can be thought of as special insertion where the inserted sequence is identical to the sequence flanking the breakpoint. Similar to insertion, the annotation of duplication may possess alternative alignment.

Example: to annotate a duplication coding region,

$ transvar canno --ccds -i 'CHD7:c.1669_1674dup'
CHD7:c.1669_1674dup  CCDS47865 (protein_coding)      CHD7    +
   chr8:g.61693564_61693569dupCCCGTC/c.1669_1674dup/p.P558_S559dupPS inside_[cds_in_exon_2]
   CSQN=InFrameInsertion;left_align_gDNA=g.61693561_61693562insTCCCCG;unalign_gD
   NA=g.61693562_61693567dupTCCCCG;left_align_cDNA=c.1668_1669insTCCCCG;unalign_
   cDNA=c.1669_1674dupTCCCCG;left_align_protein=p.H556_S557insSP;unalign_protein
   =p.S557_P558dupSP;phase=0;source=CCDS

Example: a duplication on the nucleotide level may lead to frame-shift or block substitution on the amino acid level,

$ transvar canno --ccds -i 'CHD7:c.1668_1669dup'
CHD7:c.1668_1669dup  CCDS47865 (protein_coding)      CHD7    +
   chr8:g.61693561_61693562dupTT/c.1668_1669dup/p.S557Ffs*8  inside_[cds_in_exon_2]
   CSQN=Frameshift;left_align_gDNA=g.61693560_61693561insTT;unalign_gDNA=g.61693
   561_61693562dupTT;left_align_cDNA=c.1667_1668insTT;unalign_cDNA=c.1668_1669du
   pTT;source=CCDS

Example: to annotate a duplication in intronic region,

$ transvar canno --ccds -i 'CHD7:c.1666-5_1666-3dup'
CHD7:c.1666-5_1666-3dup      CCDS47865 (protein_coding)      CHD7    +
   chr8:g.61693554_61693556dupCTC/c.1666-5_1666-3dup/.       inside_[intron_between_exon_1_and_2]
   CSQN=IntronicInsertion;left_align_gDNA=g.61693553_61693554insCTC;unalign_gDNA
   =g.61693554_61693556dupCTC;left_align_cDNA=c.1666-6_1666-5insCTC;unalign_cDNA
   =c.1666-5_1666-3dupCTC;source=CCDS