SAMtools. SAM BAM. mapping. BAM sort & indexing (ex: IGV) SNP call

Transcription

1 SAMtools SAM/BAM mapping BAM SAM BAM BAM sort & indexing (ex: IGV) mapping SNP call SAMtools NGS

2 Program: samtools (Tools for alignments in the SAM format) Version: Usage: samtools <command> [options] Command: view SAM<->BAM conversion sort sort alignment file mpileup multi-way pileup depth compute the depth faidx index/extract FASTA tview text alignment viewer index index alignment idxstats BAM index stats fixmate fix mate information flagstat simple stats calmd recalculate MD/NM tags and '=' bases merge merge sorted alignments rmdup remove PCR duplicates reheader replace BAM header cat concatenate BAMs bedcov read depth per BED region targetcut cut fosmid regions (for fosmid pool only) phase phase heterozygotes bamshuf shuffle and group alignments by name samtools view Usage: samtools view [options] <in.bam> <in.sam> [region1 [...]] Options: -b output BAM -h print header for the SAM output -H print header only (no alignments) -S input is SAM -u uncompressed BAM output (force -b) -x output FLAG in HEX (samtools-c specific) -X output FLAG in string (samtools-c specific) -c print only the count of matching records -t FILE list of reference names and lengths (force -S) [null] -T FILE reference sequence file (force -S) [null] -o FILE output file name [stdout] -R FILE list of read groups to be outputted [null] -f INT required flag, 0 for unset [0] -F INT filtering flag, 0 for unset [0] -q INT minimum mapping quality [0] -l STR only output reads in library STR [null] -r STR only output reads in read group STR [null] -? longer help

3 Q1. less ex1.sam Q2. less ex1.bam Q3. samtools Q4. samtools view samtools view Q5. samtools view ex1.bam Q6. samtools view ex1.sam bam ex1_myself.bam ex1.bam Q7. ls *Q8. samtools view -f ex1_myself.bam BAM index BAM BAM

4 Q1. samtools index samtools index Q2. ex1_myself.bam index index sort bam Q3. ex1_myself.bam sort samtools Q4. Q2 sort bam index *Q5. gn:buc > samtools view file_sorted.bam gn:buc: sam 2 Yes/No 1/ = 83 Read2 1 seq read 2 3 read Read1 2 7 Read2 : Read1 83 map

5 ref Read2 Read1 read Read1 Read *Q6.

6 flagstat, depth flagstat: Collect some statistics about alignment $ samtools flagstat NA12878.chr16p.bam in total (QC-passed reads + QC-failed reads) duplicates mapped (96.52%:nan%) paired in sequencing read read properly paired (83.33%:nan%) with itself and mate mapped singletons (4.11%:nan%) with mate mapped to a different chr with mate mapped to a different chr (mapq>=5) depth: compute the depth 1 coverage (depth) $ samtools depth NA12878.chr16p.bam head Q1. samtools flagstat depth ex1_myself.sort.bam Q2. ex1_myself.bam flagstat Q3. ex1_myself.bam depth

7 mpileup Usage: samtools mpileup [options] in1.bam [in2.bam [...]] Input options: -6 assume the quality is in the Illumina-1.3+ encoding -A count anomalous read pairs -B disable BAQ computation -b FILE list of input BAM files [null] -C INT parameter for adjusting mapq; 0 to disable [0] -d INT max per-bam depth to avoid excessive memory usage [250] -E extended BAQ for higher sensitivity but lower specificity -f FILE faidx indexed reference sequence file [null] -G FILE exclude read groups listed in FILE [null] -l FILE list of positions (chr pos) or regions (BED) [null] -M INT cap mapping quality at INT [60] -r STR region in which pileup is generated [null] -R ignore RG tags -q INT skip alignments with mapq smaller than INT [0] -Q INT skip bases with baseq/baq smaller than INT [13] Output options: -D output per-sample DP in BCF (require -g/-u) -g generate BCF output (genotype likelihoods) -O output base positions on reads (disabled by -g/-u) -s output mapping quality (disabled by -g/-u) -S output per-sample strand bias P-value in BCF (require -g/-u) -u generate uncompress BCF output SNP/INDEL genotype likelihoods options (effective with `-g' or `-u'): -e INT Phred-scaled gap extension seq error probability [20] -F FLOAT minimum fraction of gapped reads for candidates [0.002] -h INT coefficient for homopolymer errors [100] -I do not perform indel calling -L INT max per-sample depth for INDEL calling [250] -m INT minimum gapped reads for indel candidates [1] -o INT Phred-scaled gap open sequencing error probability [40] -P STR comma separated list of platforms for indels [all] Notes: Assuming diploid individuals. > samtools mpileup ex1_myself.bam gn:buc N 32 t$t$tttttttttttttttttttttttttttttt HHEHFGIDHFCH?15HHHGHIH gn:buc N 30 tttttttttttttttttttttttttttttt EHHFG@HGDHF)BHHHHHGHDHEHHHHHEG gn:buc N 30 tttttttttttttttttttttttttttttt DHGGGBH?DHF1CHHHFHGHGHFHHHHGEF gn:buc N 30 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaa EHFCFBHFDHD;5FHHGHEHDF?HHHHGDF gn:buc N 30 tttttttttttttttttttttttttttttt 6HDG=BHFDCE+;BHBFH?H?FFHGHHHEB gn:buc N 30 cccccccccccccccccccccccccccccc GGFEG8HGGHH,;HHHFHGHEGEHGHHHFH gn:buc N 30 cccccccccccccccccccccacccccccc EHEEH8HHGEE0/HDHFHDHA?FHHHHHGH gn:buc N 30 c$ccccccccccccccccccccccccccccc EHGEH<HGFHHA=HHHCHDF<FFHHHHHEH gn:buc N 29 ccccccccccccccccccccccccccccc HFCH<HFFGHCEHGHGHCHAHFHHHHH@H gn:buc N 29 t$tttttttttttttttttttttttttttt HHEE5HDEEFFEHHHFHFHF?DHHHGHFH gn:buc N 28 CCccCCCcccccCCCCcCCcCccCCCcc HFH5H?EHHG;HHHEHGHFF?HHHHHBH gn:buc N 28 CCccCCCcccccCCCCcCCcCccCCCcc HEH<HEEHHAEHHHGHGHGHEHHHHHGH gn:buc N 28 AAaaAAAaaaaaAAAAaAAaAaaAAAaa HEH5G<DHHDFHGGGGGHFHCGHHHHEH gn:buc N 28 A$AaaAAAaaaaaAAAAaAAaAaaAAAaa EBH:HEEGHDBHHHHHGHFHFHHHHHGH gn:buc N 27 T$ttTTTtttttTTTTtTTtTttTTTtt <H>HEEHF4EHHHHHHFGHFHHHHHFH

8 Q1. buc.genome.fasta mpileup Q2. index samtools faidx fasta index Q1 index gn:buc A 28,$,..,,.,.,,...,..,..,., AHHBHHHHHHEHHHDHHHDFHHHHHE<C gn:buc G 27,..,,.,.,,...,..,..,., HHEGHHHHHEHHHGFHDEFHGGHHE8< gn:buc A 28,..,,.,.,,...,..,..,.,^K, HHEHHHHHH>HHHHHHGCEHHHHH@<<< gn:buc A 28,..,,.,.,,...,..,..,.,, HG6HHHHHH:HHHHHHHDFHHGHHE>C= gn:buc A 28,..,,.,.,,...,..,..,.,, HEEGHHHHH@HHHEHHHDEHHGHGE6@> gn:buc A 29,..,,.,.,,...,..,..,.,,^K. HHEHHHHHH*HHHGHHHDFHHGDGE7/>8 gn:buc A 29,..,,.,.,,...,..,..,.,,. HEEHHHHHH@HHHEHHHD?HHGEH<6??8 gn:buc A 29,..,,.,.,,...,..,..,.,,. FHEFHHHGH8HHGHGHHDDEHFDH70CA9 gn:buc G 29 ccccccccccccccccccccccccccccc HFEHHHHHHEHHHHHHHCDEHBEH@;DB; gn:buc A 29,..,,.,.,,...,..,..,.,,. mpileup -> bcftools SAMtools BCFtools variant caller mpileup BCFtools variant vcf )

9 Q1. ex1_myself.bam vcf Q2. less vcf SAMtools tview text alignment viewer viewer fixmate fix mate information merge merge sorted alignments BAM merge rmdup remove PCR duplicates PCR duplicate

10 Q1. ex1_myself.sort.bam rmdup SAMtools SAMtools NGS NGS