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Documentation for the ANNOVAR software
http://annovar.openbioinformatics.org
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Which score is chosen when multiple prediction score are annotated with dbNSFP3.0? #73

Closed roselucia closed 5 years ago

roselucia commented 5 years ago

Hello,

in the technical comment in the section Polyphen 2 it si described, that if more than one score exists (due to multiple isoforms) only the largest score (most deleterious) is used in the annotation. How does Annovar deal with multiple scores when annotating the scores of the dataset dbnsfp30a, such as SIFT, MutationTaster, etc.? Is always the most deleterious one/the one with the highes impact used?

Thanks! Best regards, Rose

kaichop commented 5 years ago

Yes, the most deleterious one is used as the score.

On Fri, Sep 13, 2019 at 7:58 AM roselucia notifications@github.com wrote:

Hello,

in the technical comment in the section Polyphen 2 it si described, that if more than one score exists (due to multiple isoforms) only the largest score (most deleterious) is used in the annotation. How does Annovar deal with multiple scores when annotating the scores of the dataset dbnsfp30a, such as SIFT, MutationTaster, etc.? Is always the most deleterious one/the one with the highes impact used?

Thanks! Best regards, Rose

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roselucia commented 5 years ago

Many thanks! May I ask you why for the variant chr17:41244936 G>A Annovar selects the following scores? The Provean score given by the Annovar annotation: 5.84 Multiple Scores are available for the following transcript in dbNSFP3.0 database: ENST00000357654, ENST00000493795, ENST00000471181, ENST00000354071 | 5.74, 5.7, 5.84, 5.71. According to the dbNSFP3.0 readme file the most deleterious score is the smallest one, which then would not be 5.84 but 5.7. Why does Annovar annotate 5.84? So is surely always the most deleterious score used, even though when this would be the smallest score given for a variant?

MutationTaster score given by the Annovar annotation: 0.997 Multiple scores are available for the following transcript in dbNSFP3.0 database: P824L, P871L, P871L, P871L, P575L, P871L 0.996297, 0.996297, 0.996698, 0.996698, 0.996297, 0.996297 Is always the greatest p-value annotated by Annovar?

Thanks again for your help!

kaichop commented 5 years ago

I checked original code. The largest rankscore, or converted_score, or converted_rankscore are used to represent multiple scores. The original raw scores are not used. These scores are supposed to be more deleterious, if scores are higher. They are different from raw scores.

You did not give details, so I assume that you are referring to version 3.5a. In the header of 3.5a, there are PROVEAN_score PROVEAN_converted_rankscore PROVEAN_pred fields for dbNSFP, and the PROVEAN_converted_rankscore field is the one that is used in the sorting procedure to select largest score.

On Wed, Oct 2, 2019 at 9:59 AM roselucia notifications@github.com wrote:

Many thanks! May I ask you why for the variant chr17:41244936 G>A Annovar selects the following scores? The Provean score given by the Annovar annotation: 5.84 Multiple Scores are available for the following transcript in dbNSFP3.0 database: ENST00000357654, ENST00000493795, ENST00000471181, ENST00000354071 | 5.74, 5.7, 5.84, 5.71. According to the dbNSFP3.0 readme file the most deleterious score is the smallest one, which then would not be 5.84 but 5.7. Why does Annovar annotate 5.84? So is surely always the most deleterious score used, even though when this would be the smallest score given for a variant?

MutationTaster score given by the Annovar annotation: 0.997 Multiple scores are available for the following transcript in dbNSFP3.0 database: P824L, P871L, P871L, P871L, P575L, P871L 0.996297, 0.996297, 0.996698, 0.996698, 0.996297, 0.996297 Is always the greatest p-value annotated by Annovar?

Thanks again for your help!

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roselucia commented 5 years ago

Thanks for you help and sorry for the missing required information. I used the 3.0a version. However I am only left with the headers PROVEAN_score and PROVEAN_Pred. The header PROVEAN_converted_rankscore is not shown in my annotated data. Furthermore I was wondering if always the greatest p-value of the MutationTaster Score is annotated by Annovar?

1)command line argument: I tried two slightly different commands. However as expected, the result was the same

first command: perl table_annovar.pl /Users/rosefroehlich/Desktop/vcf-original/QIAGEN/smcounter2/smCounter2_neu/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.vcf humandb/ -buildver hg19 -out '/Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno' -remove -protocol refGene,ensGene,cytoBand,exac03,gnomad211_genome,gnomad211_exome,1000g2015aug_all,1000g2015aug_eur,avsnp147,dbnsfp30a,cosmic70,clinvar_20190305 -operation g,g,r,f,f,f,f,f,f,f,f,f -nastring . -vcfinput -polish

second command: perl table_annovar.pl /Users/rosefroehlich/Desktop/vcf-original/QIAGEN/smcounter2/smCounter2_neu/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.vcf humandb/ -buildver hg19 -out /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno -remove -protocol refGene,ensGene,cytoBand,exac03,gnomad211_genome,gnomad211_exome,1000g2015aug_all,1000g2015aug_eur,avsnp147,dbnsfp30a,cosmic70,clinvar_20190305 -operation g,g,r,f,f,f,f,f,f,f,f,f -nastring . -vcfinput -polish

2)error message in screen/ 3) example input file I sent you an email with a copy of the screen of my second command as well as an example input file attached.

4)Mac (We also tried it on Ubuntu and the result was the same)

Thanks for your help.

kaichop commented 5 years ago

I strongly suggest that you use the 3.5 version or 3.3 version because that's the date when the rankscores are introduced so that the most deleterious score for a variant is annotated.

I cannot see any screen message. I do not see any problem in your command line though.

roselucia commented 5 years ago

Dear Mr. Wang,

1) Do I understand you correctly that before introducing the rankscores (starting in version dbNSFP 3.3.), the highest score was chosen (e.g. for "PROVEAN_score")? 2) I annotated the GnomAD population frequencies using the v211 database. While comparing the frequencies annotated for two variants with the ones supplied on the GnomAD website, I found that the non_cancer_AF_popmax never includes a genomic frequency for the annotated. Do you have an idea what my mistake is?

The screen messages as well as an example input file I sent you via email, as I cannot attach the input file. If you still need it in order to be able to answer the questions and didn't get it, please let me know.

Below you find a copy of the screen of my second command.

Thanks for your help. Rose

„Last login: Fri Oct 4 16:15:45 on ttys001 Rose-Frohlichs-MacBook-Pro:~ rosefroehlich$ cd /Users/rosefroehlich/Desktop/ANNOVAR/annovar Rose-Frohlichs-MacBook-Pro:annovar rosefroehlich$ perl table_annovar.pl /Users/rosefroehlich/Desktop/vcf-original/QIAGEN/smcounter2/smCounter2_neu/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.vcf humandb/ -buildver hg19 -out /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno -remove -protocol refGene,ensGene,cytoBand,exac03,gnomad211_genome,gnomad211_exome,1000g2015aug_all,1000g2015aug_eur,avsnp147,dbnsfp30a,cosmic70,clinvar_20190305 -operation g,g,r,f,f,f,f,f,f,f,f,f -nastring . -vcfinput -polish

NOTICE: Running with system command <convert2annovar.pl -includeinfo -allsample -withfreq -format vcf4 /Users/rosefroehlich/Desktop/vcf-original/QIAGEN/smcounter2/smCounter2_neu/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.vcf > /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.avinput> NOTICE: Finished reading 380 lines from VCF file NOTICE: A total of 282 locus in VCF file passed QC threshold, representing 263 SNPs (188 transitions and 75 transversions) and 19 indels/substitutions NOTICE: Finished writing allele frequencies based on 263 SNP genotypes (188 transitions and 75 transversions) and 19 indels/substitutions for 1 samples

NOTICE: Running with system command <table_annovar.pl /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.avinput humandb/ -buildver hg19 -outfile /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno -remove -protocol refGene,ensGene,cytoBand,exac03,gnomad211_genome,gnomad211_exome,1000g2015aug_all,1000g2015aug_eur,avsnp147,dbnsfp30a,cosmic70,clinvar_20190305 -operation g,g,r,f,f,f,f,f,f,f,f,f -nastring . -polish -otherinfo>

NOTICE: Processing operation=g protocol=refGene

NOTICE: Running with system command <annotate_variation.pl -geneanno -buildver hg19 -dbtype refGene -outfile /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.refGene -exonsort /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.avinput humandb/> NOTICE: Output files were written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.refGene.variant_function, /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.refGene.exonic_variant_function NOTICE: Reading gene annotation from humandb/hg19_refGene.txt ... Done with 72212 transcripts (including 17527 without coding sequence annotation) for 28250 unique genes NOTICE: Processing next batch with 282 unique variants in 282 input lines NOTICE: Reading FASTA sequences from humandb/hg19_refGeneMrna.fa ... Done with 242 sequences WARNING: A total of 446 sequences will be ignored due to lack of correct ORF annotation

NOTICE: Running with system command <coding_change.pl /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.refGene.exonic_variant_function.orig humandb//hg19_refGene.txt humandb//hg19_refGeneMrna.fa -alltranscript -out /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.refGene.fa -newevf /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.refGene.exonic_variant_function>

NOTICE: Processing operation=g protocol=ensGene

NOTICE: Running with system command <annotate_variation.pl -geneanno -buildver hg19 -dbtype ensGene -outfile /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.ensGene -exonsort /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.avinput humandb/> NOTICE: Output files were written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.ensGene.variant_function, /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.ensGene.exonic_variant_function NOTICE: Reading gene annotation from humandb/hg19_ensGene.txt ... Done with 196501 transcripts (including 101155 without coding sequence annotation) for 57905 unique genes NOTICE: Processing next batch with 282 unique variants in 282 input lines NOTICE: Reading FASTA sequences from humandb/hg19_ensGeneMrna.fa ... Done with 343 sequences WARNING: A total of 6780 sequences will be ignored due to lack of correct ORF annotation

NOTICE: Running with system command <coding_change.pl /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.ensGene.exonic_variant_function.orig humandb//hg19_ensGene.txt humandb//hg19_ensGeneMrna.fa -alltranscript -out /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.ensGene.fa -newevf /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.ensGene.exonic_variant_function>

NOTICE: Processing operation=r protocol=cytoBand

NOTICE: Running with system command <annotate_variation.pl -regionanno -dbtype cytoBand -buildver hg19 -outfile /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.avinput humandb/> NOTICE: Output file is written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_cytoBand NOTICE: Reading annotation database humandb/hg19_cytoBand.txt ... Done with 862 regions NOTICE: Finished region-based annotation on 282 genetic variants

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 /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.avinput humandb/ -otherinfo> NOTICE: the --dbtype exac03 is assumed to be in generic ANNOVAR database format NOTICE: Variants matching filtering criteria are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_exac03_dropped, other variants are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_exac03_filtered NOTICE: Processing next batch with 282 unique variants in 282 input lines NOTICE: Database index loaded. Total number of bins is 749886 and the number of bins to be scanned is 258 NOTICE: Scanning filter database humandb/hg19_exac03.txt...Done

NOTICE: Processing operation=f protocol=gnomad211_genome NOTICE: Finished reading 17 column headers for '-dbtype gnomad211_genome'

NOTICE: Running system command <annotate_variation.pl -filter -dbtype gnomad211_genome -buildver hg19 -outfile /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.avinput humandb/ -otherinfo> NOTICE: the --dbtype gnomad211_genome is assumed to be in generic ANNOVAR database format NOTICE: Variants matching filtering criteria are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_gnomad211_genome_dropped, other variants are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_gnomad211_genome_filtered NOTICE: Processing next batch with 282 unique variants in 282 input lines NOTICE: Database index loaded. Total number of bins is 28119985 and the number of bins to be scanned is 259 NOTICE: Scanning filter database humandb/hg19_gnomad211_genome.txt...Done

NOTICE: Processing operation=f protocol=gnomad211_exome NOTICE: Finished reading 17 column headers for '-dbtype gnomad211_exome'

NOTICE: Running system command <annotate_variation.pl -filter -dbtype gnomad211_exome -buildver hg19 -outfile /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.avinput humandb/ -otherinfo> NOTICE: the --dbtype gnomad211_exome is assumed to be in generic ANNOVAR database format NOTICE: Variants matching filtering criteria are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_gnomad211_exome_dropped, other variants are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_gnomad211_exome_filtered NOTICE: Processing next batch with 282 unique variants in 282 input lines NOTICE: Database index loaded. Total number of bins is 773145 and the number of bins to be scanned is 258 NOTICE: Scanning filter database humandb/hg19_gnomad211_exome.txt...Done

NOTICE: Processing operation=f protocol=1000g2015aug_all

NOTICE: Running system command <annotate_variation.pl -filter -dbtype 1000g2015aug_all -buildver hg19 -outfile /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.avinput humandb/> NOTICE: Variants matching filtering criteria are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_ALL.sites.2015_08_dropped, other variants are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_ALL.sites.2015_08_filtered NOTICE: Processing next batch with 282 unique variants in 282 input lines NOTICE: Database index loaded. Total number of bins is 2824642 and the number of bins to be scanned is 217 NOTICE: Scanning filter database humandb/hg19_ALL.sites.2015_08.txt...Done

NOTICE: Processing operation=f protocol=1000g2015aug_eur

NOTICE: Running system command <annotate_variation.pl -filter -dbtype 1000g2015aug_eur -buildver hg19 -outfile /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.avinput humandb/> NOTICE: Variants matching filtering criteria are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_EUR.sites.2015_08_dropped, other variants are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_EUR.sites.2015_08_filtered NOTICE: Processing next batch with 282 unique variants in 282 input lines NOTICE: Database index loaded. Total number of bins is 2812033 and the number of bins to be scanned is 217 NOTICE: Scanning filter database humandb/hg19_EUR.sites.2015_08.txt...Done

NOTICE: Processing operation=f protocol=avsnp147

NOTICE: Running system command <annotate_variation.pl -filter -dbtype avsnp147 -buildver hg19 -outfile /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.avinput humandb/> NOTICE: Variants matching filtering criteria are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_avsnp147_dropped, other variants are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_avsnp147_filtered NOTICE: Processing next batch with 282 unique variants in 282 input lines NOTICE: Database index loaded. Total number of bins is 27868332 and the number of bins to be scanned is 259 NOTICE: Scanning filter database humandb/hg19_avsnp147.txt...Done

NOTICE: Processing operation=f protocol=dbnsfp30a NOTICE: Finished reading 34 column headers for '-dbtype dbnsfp30a'

NOTICE: Running system command <annotate_variation.pl -filter -dbtype dbnsfp30a -buildver hg19 -outfile /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.avinput humandb/ -otherinfo> NOTICE: the --dbtype dbnsfp30a is assumed to be in generic ANNOVAR database format NOTICE: Variants matching filtering criteria are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_dbnsfp30a_dropped, other variants are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_dbnsfp30a_filtered NOTICE: Processing next batch with 282 unique variants in 282 input lines NOTICE: Database index loaded. Total number of bins is 548895 and the number of bins to be scanned is 257 NOTICE: Scanning filter database humandb/hg19_dbnsfp30a.txt...Done

NOTICE: Processing operation=f protocol=cosmic70

NOTICE: Running system command <annotate_variation.pl -filter -dbtype cosmic70 -buildver hg19 -outfile /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.avinput humandb/> NOTICE: the --dbtype cosmic70 is assumed to be in generic ANNOVAR database format NOTICE: Variants matching filtering criteria are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_cosmic70_dropped, other variants are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_cosmic70_filtered NOTICE: Processing next batch with 282 unique variants in 282 input lines NOTICE: Database index loaded. Total number of bins is 232279 and the number of bins to be scanned is 210 NOTICE: Scanning filter database humandb/hg19_cosmic70.txt...Done

NOTICE: Processing operation=f protocol=clinvar_20190305 NOTICE: Finished reading 5 column headers for '-dbtype clinvar_20190305'

NOTICE: Running system command <annotate_variation.pl -filter -dbtype clinvar_20190305 -buildver hg19 -outfile /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.avinput humandb/ -otherinfo> NOTICE: the --dbtype clinvar_20190305 is assumed to be in generic ANNOVAR database format NOTICE: Variants matching filtering criteria are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_clinvar_20190305_dropped, other variants are written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_clinvar_20190305_filtered NOTICE: Processing next batch with 282 unique variants in 282 input lines NOTICE: Database index loaded. Total number of bins is 45822 and the number of bins to be scanned is 145 NOTICE: Scanning filter database humandb/hg19_clinvar_20190305.txt...Done

NOTICE: Multianno output file is written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_multianno.txt NOTICE: Reading from /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_multianno.txt

NOTICE: VCF output is written to /Users/rosefroehlich/Desktop/ANNOVAR/Annotierten_Dateien/QIAGEN/QIAseq-DNA-smCounter2.5BC_S15.smCounter.anno.hg19_multianno.hg19_multianno.vcf Rose-Frohlichs-MacBook-Pro:annovar rosefroehlich$