Am

elG

houi

la, C

laud

ia C

hica

, Em

naA

chou

ri&

Fa

tma

Gue

rfali

C3B

I Ha

nds-

on N

GS

cour

se –

IPP

–23

rdN

ov 2

016

1

SAMandBAMformats

Am

elG

houi

la, C

laud

ia C

hica

, Em

naA

chou

ri&

Fa

tma

Gue

rfali

C3B

I Ha

nds-

on N

GS

cour

se –

IPP

–23

rdN

ov 2

016

2

Rawsequencedata:Fastq files

Mapping(Bowtie,BWAorothers)

BAM/SAMfiles

• AftermappingtheFASTQfiletothereferencegenomeyouwillendupwithaSAMorBAMalignmentfile

• SAMstandsforSequenceAlignment/Mapformat

• AsingleSAMfilecanstoremapped,unmapped,andevenQC-failedreadsfromasequencingrun,andindexedtoallowrapidaccess.ThismeansthattherawsequencingdatacanbefullyrecapitulatedfromtheSAM/BAMfile.

SAM,BAMformats

Am

elG

houi

la, C

laud

ia C

hica

, Em

naA

chou

ri&

Fa

tma

Gue

rfali

C3B

I Ha

nds-

on N

GS

cour

se –

IPP

–23

rdN

ov 2

016

LiShen,2014

SAMFormat

Am

elG

houi

la, C

laud

ia C

hica

, Em

naA

chou

ri&

Fa

tma

Gue

rfali

C3B

I Ha

nds-

on N

GS

cour

se –

IPP

–23

rdN

ov 2

016

Rawsequencedata:Fastq files

Mapping(Bowtie,BWAorothers)

BAM/SAMfiles

• SAMisrarelyhelpfulandreallytakesuptoomuchspace whichiswhyweuseonlytheBAMinprinciple

• ABAMfile(.bam)isthebinaryversionofaSAMfile(savingstorageandfastermanipulation)

SAM,BAMformats

4

Am

elG

houi

la, C

laud

ia C

hica

, Em

naA

chou

ri&

Fa

tma

Gue

rfali

C3B

I Ha

nds-

on N

GS

cour

se –

IPP

–23

rdN

ov 2

016

§ ASAMfile(.sam)isatab-delimitedtextfilethatcontainssequencealignmentdata

§ SAMfilescanbeopenedusingatexteditororviewedusingtheUNIX"more"command

§ Mostalignmentprogramswillsupply:

- aheader:describingtheformatversion,sortingorderofthereads,genomicsequencestowhichthereadsweremapped

- analignmentsection:containstheinformationforeachsequenceaboutwhere/howitalignstothereferencegenome

Rawsequencedata:Fastq files

Mapping(Bowtie,BWAorothers)

BAM/SAMfiles

SAM,BAMformats

5

Am

elG

houi

la, C

laud

ia C

hica

, Em

naA

chou

ri&

Fa

tma

Gue

rfali

C3B

I Ha

nds-

on N

GS

cour

se –

IPP

–23

rdN

ov 2

016

SAM,BAMformats

Header:Alignmentsection11columns(tab-separated)

6

Am

elG

houi

la, C

laud

ia C

hica

, Em

naA

chou

ri&

Fa

tma

Gue

rfali

C3B

I Ha

nds-

on N

GS

cour

se –

IPP

–23

rdN

ov 2

016

7

SAMFormat

http://samtools.sourceforge.net/SAM1.pdfhttp://genome.sph.umich.edu/wiki/SAM

QNAME FLAG RNAME MAPQ RNEX

T

PNEX

T

TLEN

SEQPOS

CIGAR

QUAL

Am

elG

houi

la, C

laud

ia C

hica

, Em

naA

chou

ri&

Fa

tma

Gue

rfali

C3B

I Ha

nds-

on N

GS

cour

se –

IPP

–23

rdN

ov 2

016

(http://samtools.github.io/hts-specs/SAMv1.pdf)

QNAME:QuerytemplateNAME.Reads/segmentshavingidenticalQNAMEareregardedtocomefromthesametemplate.AQNAME‘*’indicatestheinformationisunavailable.

8

SAMfomat

Am

elG

houi

la, C

laud

ia C

hica

, Em

naA

chou

ri&

Fa

tma

Gue

rfali

C3B

I Ha

nds-

on N

GS

cour

se –

IPP

–23

rdN

ov 2

016

(http://samtools.github.io/hts-specs/SAMv1.pdf)

FLAG:FLAG:bitwiseFLAG(idealforcompression).

9

SAMfomat(2)

11boolean flagsallstotred inasingecolumn

Am

elG

houi

la, C

laud

ia C

hica

, Em

naA

chou

ri&

Fa

tma

Gue

rfali

C3B

I Ha

nds-

on N

GS

cour

se –

IPP

–23

rdN

ov 2

016

10

SAMfile

read mapped toposition7:FLAG163(=1+2+32+128):- Readis thesecondread inthepair(128)- Readis properly paired (1+2)- its mateis mapped to37onthereversestrand (32)

SAMflag:example

Am

elG

houi

la, C

laud

ia C

hica

, Em

naA

chou

ri&

Fa

tma

Gue

rfali

C3B

I Ha

nds-

on N

GS

cour

se –

IPP

–23

rdN

ov 2

016

Explainflagtool:https://broadinstitute.github.io/picard/explain-flags.html

11

DecodingSAMflags

Am

elG

houi

la, C

laud

ia C

hica

, Em

naA

chou

ri&

Fa

tma

Gue

rfali

C3B

I Ha

nds-

on N

GS

cour

se –

IPP

–23

rdN

ov 2

016

(http://samtools.github.io/hts-specs/SAMv1.pdf)

The MAPQvaluecanbeusedtofigureouthowuniqueanalignmentisinthegenome.ü Largenumber,>10 indicatesit'slikelythealignmentisunique.ü 255indicatesthatthemappingqualityisnotavailable

12

SAMfomat(3)

Itequals−10log10Pr{mappingpositioniswrong},roundedtothenearestinteger.

Am

elG

houi

la, C

laud

ia C

hica

, Em

naA

chou

ri&

Fa

tma

Gue

rfali

C3B

I Ha

nds-

on N

GS

cour

se –

IPP

–23

rdN

ov 2

016

• The CIGAR string is a sequence of numbers and lettersrepresenting the associated information on bases alignmentused to indicate things like which bases align (either amatch/mismatch) with the reference, are deleted from thereference, and if there are insertions that are not in the reference

SAMfomat:CIGARstring

Moreinformationabouttheseformatsavailablehere:http://samtools.sourceforge.nethttps://samtools.github.io/hts-specs/SAMv1.pdf

13

Am

elG

houi

la, C

laud

ia C

hica

, Em

naA

chou

ri&

Fa

tma

Gue

rfali

C3B

I Ha

nds-

on N

GS

cour

se –

IPP

–23

rdN

ov 2

016

Mapped andunmapped reads areimported into SAM/BAMformat

ThestandardCIGARdescriptionofpairwise alignment defines three operations:‘M’foralignment match,‘I’forinsertioncompared with thereference and‘D’fordeletion.

(NB:ThePOSindicates that theread aligns starting at position5onthereference)

TheCIGAR:3M=3basesintheread sequence align with thereference.1I=Thenext baseintheread does notexist inthereference.1D=Thereference basedoes notexist intheread sequence

POS:5CIGAR:3M1I3M1D2M

http://genome.sph.umich.edu/wiki/SAM

SAMfomat:CIGARstring

14

Am

elG

houi

la, C

laud

ia C

hica

, Em

naA

chou

ri&

Fa

tma

Gue

rfali

C3B

I Ha

nds-

on N

GS

cour

se –

IPP

–23

rdN

ov 2

016

(Lietal.,2009)

Alignments

SAMfile

Examples ofCIGARstringsfordifferent typesofalignments

SAMfomat:CIGARstring

15

Am

elG

houi

la, C

laud

ia C

hica

, Em

naA

chou

ri&

Fa

tma

Gue

rfali

C3B

I Ha

nds-

on N

GS

cour

se –

IPP

–23

rdN

ov 2

016

Nameofmate(matepairinformationforpaired-endsequencing)Positionofmate(matepairinformation)

Obviously,thechromsome andpositionareimportant.TheCIGARstringisalsoimportanttoknowwhereinsertions(i.e.introns)mightexistinyourread.

(http://samtools.github.io/hts-specs/SAMv1.pdf)

16

SAMformat(5)

SAMfomat(5)