Presentation is loading. Please wait.

Presentation is loading. Please wait.

IMGS 2012 Bioinformatics Workshop: File Formats for Next Gen Sequence Analysis.

Similar presentations


Presentation on theme: "IMGS 2012 Bioinformatics Workshop: File Formats for Next Gen Sequence Analysis."— Presentation transcript:

1 IMGS 2012 Bioinformatics Workshop: File Formats for Next Gen Sequence Analysis

2 Gigabases Cost per Kb Lucinda Fulton, The Genome Center at Washington University CostThroughput

3 Sequencing Technologies

4 Sequence “Space” Roche 454 – Flow space – Measure pyrophosphate released by a nucleotide when it is added to a growing DNA chain – Flow space describes sequence in terms of these base incorporations – AB SOLiD – Color space – Sequencing by DNA ligation via synthetic DNA molecules that contain two nested known bases with a flouorescent dye – Each base sequenced twice – Illumina/Solexa – Base space – Single base extentions of fluorescent-labeled nucleotides with protected 3 ‘ OH groups – Sequencing via cycles of base addition/detection followed deprotection of the 3’ OH – GenomeTV – Next Generation Sequencing (lecture) –

5

6

7 Flexible Good: with rapidly changing data/tech Poor: validation Human Readable Convenient for de-bugging Computer doesn’t care!

8 Sequences FASTA FASTQ SAM/BAM Alignments SAM/BAM MAF Annotations BED GTF GFF3 GVF VCF

9 FASTQ FASTA

10 FASTQ: Data Format FASTQ – Text based – Encodes sequence calls and quality scores with ASCII characters – Stores minimal information about the sequence read – 4 lines per sequence Line 1: begins followed by sequence identifier and optional description Line 2: the sequence Line 3: begins with the “+” and is followed by sequence identifiers and description (both are optional) Line 4: encoding of quality scores for the sequence in line 2 References/Documentation – – Cock et al. (2009). Nuc Acids Res 38: Sequence data format

11 FASTQ Example FASTQ example from: Cock et al. (2009). Nuc Acids Res 38: For analysis, it may be necessary to convert to the Sanger form of FASTQ.

12 FASTQ: Details FASTQ – Text based – Encodes sequence calls and quality scores with ASCII characters – Stores minimal information about the sequence read – 4 lines per sequence Line 1: begins followed by sequence identifier and optional description Line 2: the sequence Line 3: begins with the “+” and is followed by sequence identifiers and description (both are optional) Line 4: encoding of quality scores for the sequence in line 2 References/Documentation – – Cock et al. (2009). Nuc Acids Res 38:

13 Q = Phred Quality Scores P = Base-calling error probabilities Quality scores

14 !"#$%&'()*+,-./ :; | | | | | | S - Sanger Phred+33, raw reads typically (0, 40) X - Solexa Solexa+64, raw reads typically (-5, 40) I - Illumina 1.3+ Phred+64, raw reads typically (0, 40) J - Illumina 1.5+ Phred+64, raw reads typically (3, 40) with 0=unused, 1=unused, 2=Read Segment Quality Control Indicator L - Illumina 1.8+ Phred+33, raw reads typically (0, 41) Format/PlatformQualityScoreTypeASCII encoding SangerPhred: SolexaSolexa: Illumina 1.3Phred: Illumina 1.5Phred: Illumina 1.8Phred: *** Sanger format! Quality score encoding differ among the platforms Most analysis tools require Sanger fastq quality score encoding

15

16

17 SAM (Sequence Alignment/Map) SAM is the output of aligners that map reads to a reference genome – Tab delimited w/ header section and alignment section Header sections begin (are optional) Alignment section has 11 mandatory fields – BAM is the binary format of SAM Alignment data format

18 Mandatory Alignment Fields

19 Alignment Examples Alignments in SAM format CIGAR string -> 8M2I4M1D3M

20 Annotation Formats Mostly tab delimited files that describe the location of genome features (i.e., genes, etc.) Also used for displaying annotations on standard genome browsers Important for associating alignments with specific genome features descriptions Knowing format details can be important to translating results! – BED is zero based – GTF/GFF are one based

21 GTF Annotation data format

22 chr nsv chr nsv chr nsv chr nsv chr nsv chr nsv chr nsv chr chr1: chr chr1: chr chr1: chr chr1: chr chr1: chr chr1: chr chr1: chr chr1: BED format Annotation data format

23 BED: zero based, start inclusive, stop exclusive GTF/GFF: one based, inclusive Length = stop-start Length = stop-start+1

24 GRCh37 NCBI36


Download ppt "IMGS 2012 Bioinformatics Workshop: File Formats for Next Gen Sequence Analysis."

Similar presentations


Ads by Google