Annovar and the 'Otherinfo' column in VCF inputs

Question

[Garrett]

Hi all, I am relatively new to bioinformatics research and am trying to use annovar on a vcf file I inherited from another person. I am running the following line of code:

perl /project/annovar/table_annovar.pl \
  --vcfinput exome_calls_pass_step2_normalized.vcf \
  /project/annovar/humandb/ \
  -buildver hg19 \
  -out exome_calls_pass_step2_normalized_anno \
  -remove \
  -protocol refGene,genomicSuperDups,avsnp147,esp6500siv2_all,1000g2015aug_all,exac03,gnomad_exome,gnomad_genome,cadd13,dbnsfp33a,mpc,bravo, \
  -operation g,r,f,f,f,f,f,f,f,f,f,f, \
  -nastring NA \
  -otherinfo

and getting the following error output after it completely runs everything:

perl /project/annovar/table_annovar.pl --vcfinput exome_calls_pass_step2_normalized.vcf /project/annovar/humandb/hg19/ -buildver hg19 -out exome_calls_pass_step2_normalized_anno -remove -protocol refGene,genomicSuperDups,avsnp147,esp6500siv2_all,1000g2015aug_all,exac03,gnomad_exome,gnomad_genome,cadd13,dbnsfp33a,mpc,bravo, -operation g,r,f,f,f,f,f,f,f,f,f,f, -nastring .

NOTICE: Running with system command <convert2annovar.pl -includeinfo -allsample -withfreq -format vcf4 exome_calls_pass_step2_normalized.vcf >
exome_calls_pass_step2_normalized_anno.avinput>
NOTICE: Finished reading 1343550 lines from VCF file
NOTICE: A total of 1343414 locus in VCF file passed QC threshold, representing 1200272 SNPs (843271 transitions and 357001 transversions) and 143141 indels/substitutions
NOTICE: Finished writing allele frequencies based on 1309496752 SNP genotypes (920008661 transitions and 389488091 transversions) and 156166831 indels/substitutions for 1091 samples
WARNING: 1 invalid reference alleles found in input file
NOTICE: Running with system command </home/ga352/project/annovar/table_annovar.pl exome_calls_pass_step2_normalized_anno.avinput /home/ga352/project/annovar/humandb/hg19/ -buildver hg19 -outfile exome_calls_pass_step2_normalized_anno -remove -protocol refGene,genomicSuperDups,avsnp147,esp6500siv2_all,1000g2015aug_all,exac03,gnomad_exome,gnomad_genome,cadd13,dbnsfp33a,mpc,bravo, -operation g,r,f,f,f,f,f,f,f,f,f,f, -nastring . -otherinfo>
-----------------------------------------------------------------
NOTICE: Processing operation=g protocol=refGene
NOTICE: Running with system command <annotate_variation.pl -geneanno -buildver hg19 -dbtype refGene -outfile exome_calls_pass_step2_normalized_anno.refGene -exonsort exome_calls_pass_step2_normalized_anno.avinput /home/ga352/project/annovar/humandb/hg19/>
NOTICE: Output files were written to exome_calls_pass_step2_normalized_anno.refGene.variant_function, exome_calls_pass_step2_normalized_anno.refGene.exonic_variant_function
NOTICE: Reading gene annotation from /home/ga352/project/annovar/humandb/hg19/hg19_refGene.txt ... Done with 63481 transcripts (including 15216 without coding sequence annotation) for 27720 unique genes
NOTICE: Processing next batch with 1343414 unique variants in 1343414 input lines
NOTICE: Finished analyzing 1000000 query variants
NOTICE: Reading FASTA sequences from /home/ga352/project/annovar/humandb/hg19/hg19_refGeneMrna.fa ... Done with 47026 sequences
WARNING: A total of 405 sequences will be ignored due to lack of correct ORF annotation
-----------------------------------------------------------------
NOTICE: Processing operation=r protocol=genomicSuperDups
NOTICE: Running with system command <annotate_variation.pl -regionanno -dbtype genomicSuperDups -buildver hg19 -outfile exome_calls_pass_step2_normalized_anno exome_calls_pass_step2_normalized_anno.avinput /home/ga352/project/annovar/humandb/hg19/>
NOTICE: Output file is written to exome_calls_pass_step2_normalized_anno.hg19_genomicSuperDups
NOTICE: Reading annotation database /home/ga352/project/annovar/humandb/hg19/hg19_genomicSuperDups.txt ... Done with 51599 regions
NOTICE: Finished processing 1000000 variants in queryfile
NOTICE: Finished region-based annotation on 1343414 genetic variants
-----------------------------------------------------------------
NOTICE: Processing operation=f protocol=avsnp147
NOTICE: Running system command <annotate_variation.pl -filter -dbtype avsnp147 -buildver hg19 -outfile exome_calls_pass_step2_normalized_anno exome_calls_pass_step2_normalized_anno.avinput /home/ga352/project/annovar/humandb/hg19/>
NOTICE: Variants matching filtering criteria are written to exome_calls_pass_step2_normalized_anno.hg19_avsnp147_dropped, other variants are written to exome_calls_pass_step2_normalized_anno.hg19_avsnp147_filtered
NOTICE: Processing next batch with 1343414 unique variants in 1343414 input lines
NOTICE: Database index loaded. Total number of bins is 27868332 and the number of bins to be scanned is 674515
NOTICE: Scanning filter database /home/ga352/project/annovar/humandb/hg19/hg19_avsnp147.txt...Done
-----------------------------------------------------------------
NOTICE: Processing operation=f protocol=esp6500siv2_all
NOTICE: Running system command <annotate_variation.pl -filter -dbtype esp6500siv2_all -buildver hg19 -outfile exome_calls_pass_step2_normalized_anno exome_calls_pass_step2_normalized_anno.avinput /home/ga352/project/annovar/humandb/hg19/>
NOTICE: the --dbtype esp6500siv2_all is assumed to be in generic ANNOVAR database format
NOTICE: Variants matching filtering criteria are written to exome_calls_pass_step2_normalized_anno.hg19_esp6500siv2_all_dropped, other variants are written to exome_calls_pass_step2_normalized_anno.hg19_esp6500siv2_all_filtered
NOTICE: Processing next batch with 1343414 unique variants in 1343414 input lines
NOTICE: Database index loaded. Total number of bins is 594771 and the number of bins to be scanned is 429659
NOTICE: Scanning filter database /home/ga352/project/annovar/humandb/hg19/hg19_esp6500siv2_all.txt...Done
-----------------------------------------------------------------
NOTICE: Processing operation=f protocol=1000g2015aug_all 
NOTICE: Running system command <annotate_variation.pl -filter -dbtype 1000g2015aug_all -buildver hg19 -outfile exome_calls_pass_step2_normalized_anno exome_calls_pass_step2_normalized_anno.avinput /home/ga352/project/annovar/humandb/hg19/>
NOTICE: Variants matching filtering criteria are written to exome_calls_pass_step2_normalized_anno.hg19_ALL.sites.2015_08_dropped, other variants are written to exome_calls_pass_step2_normalized_anno.hg19_ALL.sites.2015_08_filtered
NOTICE: Processing next batch with 1343414 unique variants in 1343414 input lines
NOTICE: Database index loaded. Total number of bins is 2824642 and the number of bins to be scanned is 217017
NOTICE: Scanning filter database /home/ga352/project/annovar/humandb/hg19/hg19_ALL.sites.2015_08.txt...Done
-----------------------------------------------------------------
NOTICE: Processing operation=f protocol=exac03
NOTICE: Finished reading 8 column headers for '-dbtype exac03' 
NOTICE: Running system command <annotate_variation.pl -filter -dbtype exac03 -buildver hg19 -outfile exome_calls_pass_step2_normalized_anno exome_calls_pass_step2_normalized_anno.avinput /home/ga352/project/annovar/humandb/hg19/ -otherinfo>
NOTICE: the --dbtype exac03 is assumed to be in generic ANNOVAR database format
NOTICE: Variants matching filtering criteria are written to
exome_calls_pass_step2_normalized_anno.hg19_exac03_dropped, other variants are written to exome_calls_pass_step2_normalized_anno.hg19_exac03_filtered
NOTICE: Processing next batch with 1343414 unique variants in 1343414 input lines
NOTICE: Database index loaded. Total number of bins is 749886 and the number of bins to be scanned is 506971
NOTICE: Scanning filter database /home/ga352/project/annovar/humandb/hg19/hg19_exac03.txt...Done
-----------------------------------------------------------------
NOTICE: Processing operation=f protocol=gnomad_exome
NOTICE: Finished reading 9 column headers for '-dbtype gnomad_exome'
NOTICE: Running system command <annotate_variation.pl -filter -dbtype gnomad_exome -buildver hg19 -outfile exome_calls_pass_step2_normalized_anno exome_calls_pass_step2_normalized_anno.avinput /home/ga352/project/annovar/humandb/hg19/ -otherinfo>
NOTICE: the --dbtype gnomad_exome is assumed to be in generic ANNOVAR database format
NOTICE: Variants matching filtering criteria are written to exome_calls_pass_step2_normalized_anno.hg19_gnomad_exome_dropped, other variants are written to exome_calls_pass_step2_normalized_anno.hg19_gnomad_exome_filtered
NOTICE: Processing next batch with 1343414 unique variants in 1343414 input lines
NOTICE: Database index loaded. Total number of bins is 772860 and the number of bins to be scanned is 515235
NOTICE: Scanning filter database /home/ga352/project/annovar/humandb/hg19/hg19_gnomad_exome.txt...Done
-----------------------------------------------------------------
NOTICE: Processing operation=f protocol=gnomad_genome
NOTICE: Finished reading 8 column headers for '-dbtype gnomad_genome'
NOTICE: Running system command <annotate_variation.pl -filter -dbtype gnomad_genome -buildver hg19 -outfile exome_calls_pass_step2_normalized_anno exome_calls_pass_step2_normalized_anno.avinput /home/ga352/project/annovar/humandb/hg19/ -otherinfo>
NOTICE: the --dbtype gnomad_genome is assumed to be in generic ANNOVAR database format
NOTICE: Variants matching filtering criteria are written to exome_calls_pass_step2_normalized_anno.hg19_gnomad_genome_dropped, other variants are written to exome_calls_pass_step2_normalized_anno.hg19_gnomad_genome_filtered
NOTICE: Processing next batch with 1343414 unique variants in 1343414 input lines
NOTICE: Database index loaded. Total number of bins is 28127612 and the number of bins to be scanned is 674517
NOTICE: Scanning filter database /home/ga352/project/annovar/humandb/hg19/hg19_gnomad_genome.txt...Done
-----------------------------------------------------------------
NOTICE: Processing operation=f protocol=cadd13
NOTICE: Finished reading 2 column headers for '-dbtype cadd13'
NOTICE: Running system command <annotate_variation.pl -filter -dbtype cadd13 -buildver hg19 -outfile exome_calls_pass_step2_normalized_anno exome_calls_pass_step2_normalized_anno.avinput /home/ga352/project/annovar/humandb/hg19/ -otherinfo>
NOTICE: the --dbtype cadd13 is assumed to be in generic ANNOVAR database format
NOTICE: Variants matching filtering criteria are written to exome_calls_pass_step2_normalized_anno.hg19_cadd13_dropped, other variants are written to exome_calls_pass_step2_normalized_anno.hg19_cadd13_filtered
NOTICE: Processing next batch with 1343414 unique variants in 1343414 input lines
NOTICE: Database index loaded. Total number of bins is 28648145 and the number of bins to be scanned is 675381
NOTICE: Scanning filter database /home/ga352/project/annovar/humandb/hg19/hg19_cadd13.txt...Done
-----------------------------------------------------------------
NOTICE: Processing operation=f protocol=dbnsfp33a
NOTICE: Finished reading 66 column headers for '-dbtype dbnsfp33a'
NOTICE: Running system command <annotate_variation.pl -filter -dbtype dbnsfp33a -buildver hg19 -outfile exome_calls_pass_step2_normalized_anno exome_calls_pass_step2_normalized_anno.avinput /home/ga352/project/annovar/humandb/hg19/ -otherinfo>
NOTICE: the --dbtype dbnsfp33a is assumed to be in generic ANNOVAR database format
NOTICE: Variants matching filtering criteria are written to exome_calls_pass_step2_normalized_anno.hg19_dbnsfp33a_dropped, other variants are written to exome_calls_pass_step2_normalized_anno.hg19_dbnsfp33a_filtered
NOTICE: Processing next batch with 1343414 unique variants in 1343414 input lines
NOTICE: Database index loaded. Total number of bins is 550512 and the number of bins to be scanned is 377848
NOTICE: Scanning filter database /home/ga352/project/annovar/humandb/hg19/hg19_dbnsfp33a.txt...Done
-----------------------------------------------------------------
NOTICE: Processing operation=f protocol=mpc
NOTICE: Finished reading 1 column headers for '-dbtype mpc'
NOTICE: Running system command <annotate_variation.pl -filter -dbtype mpc -buildver hg19 -outfile exome_calls_pass_step2_normalized_anno exome_calls_pass_step2_normalized_anno.avinput /home/ga352/project/annovar/humandb/hg19/ -otherinfo>
NOTICE: the --dbtype mpc is assumed to be in generic ANNOVAR database format
NOTICE: Variants matching filtering criteria are written to exome_calls_pass_step2_normalized_anno.hg19_mpc_dropped, other variants are written to exome_calls_pass_step2_normalized_anno.hg19_mpc_filtered
NOTICE: Processing next batch with 1343414 unique variants in 1343414 input lines
NOTICE: Scanning filter database /home/ga352/project/annovar/humandb/hg19/hg19_mpc.txt...Done
-----------------------------------------------------------------
NOTICE: Processing operation=f protocol=bravo
NOTICE: Finished reading 1 column headers for '-dbtype bravo'
NOTICE: Running system command <annotate_variation.pl -filter -dbtype bravo -buildver hg19 -outfile exome_calls_pass_step2_normalized_anno exome_calls_pass_step2_normalized_anno.avinput /home/ga352/project/annovar/humandb/hg19/ -otherinfo>
NOTICE: the --dbtype bravo is assumed to be in generic ANNOVAR database format
NOTICE: Variants matching filtering criteria are written to exome_calls_pass_step2_normalized_anno.hg19_bravo_dropped, other variants are written to exome_calls_pass_step2_normalized_anno.hg19_bravo_filtered
NOTICE: Processing next batch with 1343414 unique variants in 1343414 input lines
NOTICE: Database index loaded. Total number of bins is 2822209 and the number of bins to be scanned is 216806
NOTICE: Scanning filter database /home/ga352/project/annovar/humandb/hg19/hg19_bravo.txt...Done
-----------------------------------------------------------------
NOTICE: Multianno output file is written to exome_calls_pass_step2_normalized_anno.hg19_multianno.txt
NOTICE: Reading from exome_calls_pass_step2_normalized_anno.hg19_multianno.txt Error: the last column in header row should be 'Otherinfo'

I have tried also removing the -otherinfo flag and had the same error. Looking at the structure of the vcf there does not appear to be an otherinfo column. does anyone have any suggestions as to what is the problem here and how I can fix it? Thank you so much!

[tayebeh.ranjbar1992]

Hello, I also got the same error as you. " Error: the last column in header row should start with 'Otherinfo" Did you manage to fix this error? Can you tell me how to fix this error please?

Answer 1

[kangdong318]

The problem may be of the files generated in mutect2 have a different number of columns than the header, resulting in Error: the last column in header row should be 'Otherinfo in annovar. You can find the row with a different number of columns and delete it, or add Otherinfo to the header for the maximum number of rows.

[kangdong318]

also maybe caused by input file not found.