1. Pindel user manual Kai Ye k.ye@lumc.nl

2. Preparation of Pindel input Alignment BAM file generated by BWA Alignment BAM file generated by other aligners Pindel input with sample tag (1)bam2pindel.pl Adaptor.pm (2) sam2pindel.cpp Filtered Pindel input with sample tag (3) FilterPindelReads.cpp Merge Pindel input files for paired or population sequence data

3. (1) bam2pindel.pl Written by Keiran Raine at Sanger Institute (kr2@sanger.ac.uk)kr2@sanger.ac.uk This tool was designed for BWA based BAM/SAM Illumina data You must prepare a name sorted bam file Set BAM_2_PINDEL_ADAPT setenv BAM_2_PINDEL_ADAPT Arguments: -i|input: Input BAM file (req) -o|output: Output ready for pindel -s|sample: Sample or label (sampA,sampB...) (req) -pi|insert: Required if BAM file does not have PI tag in header RG record -r|restrict: Restrict to chromosome xx Example:./bam2pindel_bwa.pl –i NameSorted.bam –o output_prefix -s tumour –om –pi 300

4. (2) sam2pindel.cpp Written by Kai Ye at Leiden University Medical Center (k.ye@lumc.nl) This tool was designed for all BAM/SAM Illumina data You must first compile the cpp source code: g++ sam2pindel.cpp –o sam2pindel –O3 5 arguments are required by sam2pindel – 1. Input sam file. – 2. Output for pindel. – 3. insert size. – 4. tag. – 5. number of extra lines (not start with @) in the beginning of the file. If you start with standard sam file (Input.sam with insert size 300)./sam2pindel Input.samOutput4Pindel.txt 300tumour0 If you start with bam file./samtools view Input.bam |./sam2pindel - Output4Pindel.txt 300 tumour0

5. Running Pindel 1. Input: the reference genome sequences in fasta format; 2. Input: the unmapped reads in a modified fastq format; 3. Output folder 4. Which chr/fragment 5. BreakDancer result: Format per line: ChrA LocA stringA ChrB LocB stringB others If you don't have BreakDancer result, please provide an empty file here. Example:. /pindelhg19.fapindel_input_chr1.txtOutput_Folderchr1empty

6. Input format of Pindel @9113 TGGGGACCGGTGGAATGCTTCCACTGGCTGGGGGGC + chr2 41149518 50 Tumor ref AnchorAnchor Strand, chr, 3’ coordinate and mapping quality of the mapped reads; sample tag

7. 18 May 20157 Output format: deletions 1base - 1million bases

8. Allow mismatches to accommodate sequence errors and SNPs D 10 ChrID 13 BP 32913041 32913052 AAATCAACTAGTGACCTTCCAGGGACAACCCGAACGTGATGAAAAGATCAaagaacctacTCTATTGGGTTTTCATACAGCTAGCGGGAAAAAAGTTAAAATTGCAAAGGAATCTTTGGACAAAGT GATGAAAAGATCA TCTGTTGGGTTTTCATACAGCTAGCGGGAAAAAAGTTAAAATTGCAAAGGAATCTTTGGACAA CAACCCGAACGTGATGAAAAGATCA TCTGTTGGGTTTTCATACAGCTAGCGGGAAAAAAGTTAAAATTGCAAAGGA CGTGATGAAAAGATCA TCTGTTGGGTTTTCATACAGCTAGCGGGAAAAAAGTTAAAATTGCAAAGGAATCTTTGGA TGAAAAGATCA TCTGTTGGGTTTTCATACAGCTAGCGGGAAAAAAGTTAAAATTGCAAAGGAATCTTTGGACAAAG GTGATGAAAAGATCA TCTGTTGGGTTTTCATACAGCTAGCGGGAAAAAAGTTAAAATTGCAAAGGAATCTTTGGAC TAGTGACCTTCCAGGGACAACCCGAACGTGATGAAAAGATCA TCTGTTGGGTTTTCATACAGCTAGCGGGAAAAAA CCTTCCAGGGACAACCCGAACGTGATGAAAAGATCA TCTGTTGGGTTTTCATACAGCTAGCGGGAAAAAAGTTAAA ACAACCCGAACGTGATGAAAAGATCA TCTGTTGGGTTTTCATACAGCTAGCGGGAAAAAAGTTAAAATTGCAAAGG CGAACGTGATGAAAAGATCA TCTGTTGGGTTTTCATACAGCTAGCGGGAAAAAAGTTAAAATTGCAAAGGAATCTT CCCGAACGTGATGAAAAGATCA TCTGTTGGGTTTTCATACAGCTAGCGGGAAAAAAGTTAAAATTGCAAAGGAATC AACCCGAACGTGATGAAAAGATCA TCTGTTGGGTTTTCATACAGCTAGCGGGAAAAAAGTTAAAATTGCAAAGGAA TGATGAAAAGATCA TCTGTTGGGTTTTCATACAGCTAGCGGGAAAAAAGTTAAAATTGCAAAGGAATCTTTGGACA ACCTTCCAGGGACAACCCGAACGTGATGAAAAGATCA TCTGTTGGGTTTTCATACAGCTAGCGGGAAAAAAGTTAA GATGAAAAGATCA TCTGTTGGGTTTTCATACAGCTAGCGGGAAAAAAGTTAAAATTGCAAAGGAATCTTTGGACAA AACCCGAACGTGATGAAAAGATCA TCTGTTGGGTTTTCATACAGCTAGCGGGAAAAAAGTTAAAATTGCAAAGGAA GAACGTGATGAAAAGATCA TCTGTTGGGTTTTCATACAGCTAGCGGGAAAAAAGTTAAAATTGCAAAGGAATCTTT 8

9. Inversions 9 sample ref

10. Large insertions 10

11. Non-template sequence in deletions, inversions and tandem duplications 11 ref sample

12. Non-template sequence: deletion of 4 bases with 2 bases inserted D 4 I 2 ChrID 3 BP 156978978 156978983 Supports 12 + 0 - 12 S1 13 SUM_MS 627 NumSupSamples 1 HCC1599a 12 CATGGCTGACTTATAAATCCCTACAGATATGTGGTTACTTCTCTACTTTCCCTTTCTTTGGCTTGGGCAACTGCCACGTTGATGCACTGGAGCCATTCTTCTGCATTCTTCTCATCCTTGGCCTTAAAGACATAGGTTTTATTGTC TTATAAATCCCTACAGATATGTGGTTACTTCTCTACTTTCCCTTTCTTTGCCTTGGGCAACTGCCAAA GATGCACT ATGTGGTTACTTCTCTACTTTCCCTTTCTTTGGCTTGGGCAACTGCCAAA GATGCACTGGAGCCATTCTTCTGCAT CTCTACTTTCCCTTTCTTTGGCTTGGGCAACTGCCAAA GATGCACTGGAGCCATTCTTCTGCATTCTTCTCATCCT AGATATGTGGTTACTTCTCTACTTTCCCTTTCTTTGGCTTGGGCAACTGCCAAA GATGCACTGGAGCCATTCTTCT TTTCCCTTTCTTTGGCTTGGGCAACTGCCAAA GATGCACTGGAGCCATTCTTCTGCATTCTTCTCATCCTTGGCCT TTCCCTTTCTTTGGCTTGGGCAACTGCCAAA GATGCACTGGAGCCATTCTTCTGCATTCTTCTCATCCTTGGCCTT TTACTTCTCTACTTTCCCTTTCTTTGGCTTGGGCAACTGCCAAA GATGCACTGGAGCCATTCTTCTGCATTCTTCT CTTGGGCAACTGCCAAA GATGCACTGGAGCCATTCTTCTGCATTCTTCTCATCCTTGGCCTTAAAGACATAGGTTT CTACAGATATGTGGTTACTTCTCTACTTTCCCTTTCTTTGGCTTGGGCAACTGCCAAA GATGCACTGGAGCCATTC AAATCCCTACAGATATGTGGTTACTTCTCTACTTTCCCTTTCTTTGGCTTGGGCAACTGCCAAA GATGCACTGGAG CTTGGGCAACTGCCAAA GATGCACTGGAGCCATTCTTCTGCATTCTTCTCATCCTTGGCCTTAAAGACATAGGTTT TTCCCTTTCTTTGGCTTGGGCAACTGCCAAA GATGCACTGGAGCCATTCTTCTGCATTCTTCTCATCCTTGGCCTT