This document discusses analyzing exome sequencing data using KNIME. It describes analyzing two exomes: 1) for a given mutation in one sample, expecting the mutation to not be present in the other sample, and 2) for composite heterozygous mutations present in both samples within the same gene. The workflow details include reading the data files, filtering and processing the variants, merging data from the two samples, and visualizing the results. Commands for implementing similar analyses via UNIX bash scripts are also provided.

1. Pierre Lindenbaum PhD UMR915 – Institut du thorax Nantes, France @yokofakun http://plindenbaum.blogspot.com [email_address] Analysing Exome Data with KNIME

2. 2 exomes sequenced

3. [m/m] 1 st case: for a given mutation we expect... not( [m/m] )

4. Files

5. $1 Position.hg19 : 142653 $2 chrom : chr10 $3 sample.ID : sample1 $4 rs.name : rsXXXX $5 hapmap_ref_other : $6 X1000Genome.obs : $7 X1000Genome.desc : $8 Freq.HTZ.ExomesV1 : 0 $9 Freq.Hom.ExomesV1 : 0 $10 A : 0 $11 C : 5 $12 G : 0 $13 T : 3 $14 modified_call : CT $15 total : 9 $16 used : 8 $17 score : 18.30:12.00 $18 reference : C $19 type : SNP_het1 $20 Gene.name : ROXAN $21 Gene.start : 143652 $22 Gene.end : 293700 $23 strand : - $24 nbre.exon : 11 $25 refseq : NR_0090 $26 typeannot : 3-UTR $27 type.pos : $28 index.cdna : $29 index.prot : $30 Taille.cdna : 1769 $31 Intron.start : $32 Intron.end : $33 codon.wild : $34 aa.wild : $35 codon.mut : $36 aa.mut : $37 cds.wild : $38 cds.mut : $39 prot.wild : $40 prot.mut : $41 mirna : no $42 region.splice : !N/A 42 columns

6. $1 Position.hg19 : 142653 $2 chrom : chr10 $3 sample.ID : sample1 $4 rs.name : rsXXXX $5 hapmap_ref_other : $6 X1000Genome.obs : $7 X1000Genome.desc : $8 Freq.HTZ.ExomesV1 : 0 $9 Freq.Hom.ExomesV1 : 0 $10 A : 0 $11 C : 5 $12 G : 0 $13 T : 3 $14 modified_call : CT $15 total : 9 $16 used : 8 $17 score : 18.30:12.00 $18 reference : C $19 type : SNP_het1 $20 Gene.name : ROXAN $21 Gene.start : 143652 $22 Gene.end : 293700 $23 strand : - $24 nbre.exon : 11 $25 refseq : NR_009 $26 typeannot : 3-UTR $27 type.pos : $28 index.cdna : $29 index.prot : $30 Taille.cdna : 1769 $31 Intron.start : $32 Intron.end : $33 codon.wild : $34 aa.wild : $35 codon.mut : $36 aa.mut : $37 cds.wild : $38 cds.mut : $39 prot.wild : $40 prot.mut : $41 mirna : no $42 region.splice : !N/A Genomic Position

7. $1 Position.hg19 : 142653 $2 chrom : chr10 $3 sample.ID : sample1 $4 rs.name : rsXXXX $5 hapmap_ref_other : $6 X1000Genome.obs : $7 X1000Genome.desc : $8 Freq.HTZ.ExomesV1 : 0 $9 Freq.Hom.ExomesV1 : 0 $10 A : 0 $11 C : 5 $12 G : 0 $13 T : 3 $14 modified_call : CT $15 total : 9 $16 used : 8 $17 score : 18.30:12.00 $18 reference : C $19 type : SNP_het1 $20 Gene.name : ROXAN $21 Gene.start : 143652 $22 Gene.end : 293700 $23 strand : - $24 nbre.exon : 11 $25 refseq : NR_0090 $26 typeannot : 3-UTR $27 type.pos : $28 index.cdna : $29 index.prot : $30 Taille.cdna : 1769 $31 Intron.start : $32 Intron.end : $33 codon.wild : $34 aa.wild : $35 codon.mut : $36 aa.mut : $37 cds.wild : $38 cds.mut : $39 prot.wild : $40 prot.mut : $41 mirna : no $42 region.splice : !N/A Sample Name

8. $1 Position.hg19 : 142653 $2 chrom : chr10 $3 sample.ID : sample1 $4 rs.name : rsXXXX $5 hapmap_ref_other : $6 X1000Genome.obs : $7 X1000Genome.desc : $8 Freq.HTZ.ExomesV1 : 0 $9 Freq.Hom.ExomesV1 : 0 $10 A : 0 $11 C : 5 $12 G : 0 $13 T : 3 $14 modified_call : CT $15 total : 9 $16 used : 8 $17 score : 18.30:12.00 $18 reference : C $19 type : SNP_het1 $20 Gene.name : ROXAN $21 Gene.start : 143652 $22 Gene.end : 293700 $23 strand : - $24 nbre.exon : 11 $25 refseq : NR_0090 $26 typeannot : 3-UTR $27 type.pos : $28 index.cdna : $29 index.prot : $30 Taille.cdna : 1769 $31 Intron.start : $32 Intron.end : $33 codon.wild : $34 aa.wild : $35 codon.mut : $36 aa.mut : $37 cds.wild : $38 cds.mut : $39 prot.wild : $40 prot.mut : $41 mirna : no $42 region.splice : !N/A RS## number

9. $1 Position.hg19 : 142653 $2 chrom : chr10 $3 sample.ID : sample1 $4 rs.name : rsXXXX $5 hapmap_ref_other : $6 X1000Genome.obs : $7 X1000Genome.desc : $8 Freq.HTZ.ExomesV1 : 0 $9 Freq.Hom.ExomesV1 : 0 $10 A : 0 $11 C : 5 $12 G : 0 $13 T : 3 $14 modified_call : CT $15 total : 9 $16 used : 8 $17 score : 18.30:12.00 $18 reference : C $19 type : SNP_het1 $20 Gene.name : ROXAN $21 Gene.start : 143652 $22 Gene.end : 293700 $23 strand : - $24 nbre.exon : 11 $25 refseq : NR_0090 $26 typeannot : 3-UTR $27 type.pos : $28 index.cdna : $29 index.prot : $30 Taille.cdna : 1769 $31 Intron.start : $32 Intron.end : $33 codon.wild : $34 aa.wild : $35 codon.mut : $36 aa.mut : $37 cds.wild : $38 cds.mut : $39 prot.wild : $40 prot.mut : $41 mirna : no $42 region.splice : !N/A Ref. & Alt. alleles

10. $1 Position.hg19 : 142653 $2 chrom : chr10 $3 sample.ID : sample1 $4 rs.name : rsXXXX $5 hapmap_ref_other : $6 X1000Genome.obs : $7 X1000Genome.desc : $8 Freq.HTZ.ExomesV1 : 0 $9 Freq.Hom.ExomesV1 : 0 $10 A : 0 $11 C : 5 $12 G : 0 $13 T : 3 $14 modified_call : CT $15 total : 9 $16 used : 8 $17 score : 18.30:12.00 $18 reference : C $19 type : SNP_het1 $20 Gene.name : ROXAN $21 Gene.start : 143652 $22 Gene.end : 293700 $23 strand : - $24 nbre.exon : 11 $25 refseq : NR_0090 $26 typeannot : 3-UTR $27 type.pos : $28 index.cdna : $29 index.prot : $30 Taille.cdna : 1769 $31 Intron.start : $32 Intron.end : $33 codon.wild : $34 aa.wild : $35 codon.mut : $36 aa.mut : $37 cds.wild : $38 cds.mut : $39 prot.wild : $40 prot.mut : $41 mirna : no $42 region.splice : !N/A Gene

11. $1 Position.hg19 : 142653 $2 chrom : chr10 $3 sample.ID : sample1 $4 rs.name : rsXXXX $5 hapmap_ref_other : $6 X1000Genome.obs : $7 X1000Genome.desc : $8 Freq.HTZ.ExomesV1 : 0 $9 Freq.Hom.ExomesV1 : 0 $10 A : 0 $11 C : 5 $12 G : 0 $13 T : 3 $14 modified_call : CT $15 total : 9 $16 used : 8 $17 score : 18.30:12.00 $18 reference : C $19 type : SNP_het1 $20 Gene.name : ROXAN $21 Gene.start : 143652 $22 Gene.end : 293700 $23 strand : - $24 nbre.exon : 11 $25 refseq : NR_0090 $26 typeannot : 3-UTR $27 type.pos : $28 index.cdna : $29 index.prot : $30 Taille.cdna : 1769 $31 Intron.start : $32 Intron.end : $33 codon.wild : $34 aa.wild : $35 codon.mut : $36 aa.mut : $37 cds.wild : $38 cds.mut : $39 prot.wild : $40 prot.mut : $41 mirna : no $42 region.splice : !N/A Prediction

12. $1 Position.hg19 : 142653 $2 chrom : chr10 $3 sample.ID : sample1 $4 rs.name : rsXXXX $5 hapmap_ref_other : $6 X1000Genome.obs : $7 X1000Genome.desc : $8 Freq.HTZ.ExomesV1 : 0 $9 Freq.Hom.ExomesV1 : 0 $10 A : 0 $11 C : 5 $12 G : 0 $13 T : 3 $14 modified_call : CT $15 total : 9 $16 used : 8 $17 score : 18.30:12.00 $18 reference : C $19 type : SNP_het1 $20 Gene.name : ROXAN $21 Gene.start : 143652 $22 Gene.end : 293700 $23 strand : - $24 nbre.exon : 11 $25 refseq : NR_0090 $26 typeannot : 3-UTR $27 type.pos : $28 index.cdna : $29 index.prot : $30 Taille.cdna : 1769 $31 Intron.start : $32 Intron.end : $33 codon.wild : $34 aa.wild : $35 codon.mut : $36 aa.mut : $37 cds.wild : $38 cds.mut : $39 prot.wild : $40 prot.mut : $41 mirna : no $42 region.splice : !N/A Homo/Hetero zygote

13. http://www.knime.org

15. Our workflow:

17. Read the data

18. Rename both “ Sample” Columns

19. Remove the sequences (save memory/speed)

20. Expect “not (snp_diff.*)” for

21. Expect “snp_diff.*” for

22. Merge data. Two columns “ SAMPLE_WILD” & “ SAMPLE_MUTATED”

23. Highlight low quality

24. Remove low quality

25. Must be in located in a Gene

26. Remove if known rs#

27. Remove if synonymous mutation

28. Remove wild allele from Alt. (cleanup)

29. Group by Gene

31. Keep mutations carried by both samples

32. Group by Gene Name & Visualize

34. Retrieve the SNPs for each Gene.

36. bash version... #remove rs #in gene #remove the low qualities #keep SNP_diff #only the non-synonymous or stop #remove DNA & prot sequences #order by GENE gunzip -c AllChrom.exome.snp.pool.new.annotation.gz |awk -F ' ' '{if(substr($4,1,2)!="rs") print;}' |awk -F ' ' '{if($20!="") print;}' |awk -F ' ' '{if(index($19,"douteux")==0) print;}' |awk -F ' ' '{if(index($19,"_diff")!=0) print;}' |awk -F ' ' '{if(index($26,"nonsense")!=0 || index($26,"missense")!=0) print;}' |cut -d ' ' -f 1-27 |sort -t ' ' -k20,20 > _jeter1.txt #extract wild exome #remove rs #remove SNP_diff #in gene #order by gene gunzip -c AllChrom.exome.snp.u2437.new.annotation.gz |awk -F ' ' '{if(substr($4,1,2)!="rs") print;}' |awk -F ' ' '{if(index($19,"douteux")==0) print;}' |awk -F ' ' '{if(index($19,"_diff")==0) print;}' |awk -F ' ' '{if($20!="") print;}' |cut -d ' ' -f 1-27 |sort -t ' ' -k20,20 > _jeter3.txt #join wild & mutated data by gene #check wild sample has no mutation in the pair of mutated snps #remove wild data join -t ' ' -1 20 -2 20 _jeter1.txt _jeter3.txt |awk -F ' ' '{if($3==$29 && int($2) == int($28) ) print;}' |cut -d ' ' -f 1 |sort | uniq rm _jeter*.txt

37. In one gene: SNP1: [m/+] SNP2: [m/+] 2 nd case: Composite heterozygous

38. The workflow:

39. Read [m] & [+] files Mutated sample Wild sample

40. Remove cDNA & protein sequences

41. Remove the SNPs having a rs#

42. Keep the heterozygous mutations

43. Remove poor quality

44. Keep the non-synonymous mutations

45. Create a new column: = chrom+”_”+position;

46. Rename the columns 'sample-id' (will generate two distinct columns after joining)

47. Left join on the column 'chrom_col'

48. Keep the mutations that were NOT part of the wild sample.

49. Cleanup, remove some columns.

50. Duplicate the table to Create two lists of SNPs (5' & 3').

51. Join both tables on gene name.

52. Keep the SNPs having: pos(snp1) < pos(snp2)

53. Display the results

54. #remove rs #only keep the 'SNP_het' #remove the low qualities #remove SNP_het* #only the non-synonymous or stop #remove DNA & prot sequences #add chrom_position flag #sort gunzip -c AllChrom.exome.snp.pool.new.annotation.gz |awk -F ' ' '{if(substr($4,1,2)!=&quot;rs&quot;) print;}' |awk -F ' ' '{if(index($19,&quot;douteux&quot;)==0) print;}' |awk -F ' ' '{if(index($19,&quot;_het&quot;)!=0) print;}' |awk -F ' ' '{if(index($26,&quot;nonsense&quot;)!=0 || index($26,&quot;missense&quot;)!=0) print;}' |cut -d ' ' -f 1-27 |awk -F ' ' '{printf(&quot;%s_%s%s&quot;,$2,$1,$0);}' |sort -t ' ' -k1,1 > _jeter1.txt #get all distinct chrom_pos in file cut -d ' ' -f 1 _jeter1.txt | sort -t ' ' -k1,1 | uniq > _jeter2.txt #extract wild exome #keep chrom,position #add chrom_position flag #sort gunzip -c AllChrom.exome.snp.u2437.new.annotation.gz |cut -d ' ' -f 1,2 |awk -F ' ' '{printf(&quot;%s_%s&quot;,$2,$1);}' |sort -t ' ' -k 1,1 | uniq > _jeter3.txt #get [m] chrom_pos not in [+] chrom_pos set comm -2 -3 _jeter2.txt _jeter3.txt > _jeter4.txt #join uniq [m] chrom_pos & mutated data #remove chrom_pos #order by gene join -t ' ' --check-order -1 1 -2 1 _jeter1.txt _jeter4.txt|cut -d ' ' -f 2- |sort -t ' ' -k 20 > _jeter5.txt #join to self using key= &quot;gene name&quot; #only keep if first mutation in same gene/chromosome and pos1< pos2 #keep some columns join -t ' ' -j 20 _jeter5.txt _jeter5.txt |awk -F ' ' '{if($3==$29 && int($2) < int($28) ) print;}' |cut -d ' ' -f 1,2,3,20,26,28,46,52 > _jeter6.txt #extract gene names cut -d ' ' -f 1 _jeter6.txt | sort | uniq rm _jeter[12345].txt bash version...

55. Last step... http://en.wikipedia.org/wiki/File:Nobel_Prize.png

56. Thanks. Remember: you should learn how to use the Unix command line...