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IMGS 2012 Bioinformatics Workshop: File Formats for Next Gen Sequence Analysis. Cost. Throughput. Gigabases. Cost per Kb. Lucinda Fulton, The Genome Center at Washington University. Sequencing Technologies. http://www.geospiza.com/finchtalk/uploaded_images/plates-and-slides-718301.png.

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Imgs 2012 bioinformatics workshop file formats for next gen sequence analysis

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


Cost

Throughput

Gigabases

Cost per Kb

Lucinda Fulton, The Genome Center at Washington University


Sequencing technologies
Sequencing Technologies

http://www.geospiza.com/finchtalk/uploaded_images/plates-and-slides-718301.png


Sequence space
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

    • http://www.youtube.com/watch?v=bFNjxKHP8Jc

  • 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

    • http://www.youtube.com/watch?v=nlvyF8bFDwM&feature=related

  • 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

    • http://www.youtube.com/watch?v=77r5p8IBwJk&feature=related

  • GenomeTV – Next Generation Sequencing (lecture)

    • http://www.youtube.com/watch?v=g0vGrNjpyA8&feature=related

http://finchtalk.geospiza.com/2008/03/color-space-flow-space-sequence-space_23.html


Flexible

Good: with rapidly changing data/tech

Poor: validation

Human Readable

Convenient for de-bugging

Computer doesn’t care!


Sequences

FASTA

FASTQ

SAM/BAM

Alignments

SAM/BAM

MAF

Annotations

BED

GTF

GFF3

GVF

VCF

http://genome.ucsc.edu/FAQ/FAQformat.html

http://www.sequenceontology.org/


Fastq

FASTA

FASTQ


Fastq data format
FASTQ: Data Format

Sequence 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 with @; 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

    • http://maq.sourceforge.net/fastq.shtml

    • Cock et al. (2009). Nuc Acids Res 38:1767-1771.


Fastq example
FASTQ Example

For analysis, it may be necessary to convert to the Sanger form of FASTQ.

  • FASTQ example from: Cock et al. (2009). Nuc Acids Res 38:1767-1771.


Fastq details
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 with @; 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

    • http://maq.sourceforge.net/fastq.shtml

    • Cock et al. (2009). Nuc Acids Res 38:1767-1771.


Quality scores

Q = Phred Quality Scores

P = Base-calling error probabilities


Quality score encoding differ among the platforms

  • !"#$%&'()*+,-./0123456789:;<=>[email protected][\]^_`abcdefghijklmnopqrstuvwxyz{|}~

  • | | | | | |

  • 33 59 64 73 104 126

    • 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/Platform QualityScoreType ASCII encoding

Sanger Phred: 0-93 33-126

SolexaSolexa:-5-62 64-126

Illumina 1.3 Phred: 0-62 64-126

Illumina 1.5 Phred: 0-62 64-126

Illumina 1.8 Phred: 0-62 33-126 *** Sanger format!

Most analysis tools require Sanger fastq quality score encoding



Sam sequence alignment map
SAM (Sequence Alignment/Map)

Alignment data format

  • SAM is the output of aligners that map reads to a reference genome

    • Tab delimited w/ header section and alignment section

      • Header sections begin with @ (are optional)

      • Alignment section has 11 mandatory fields

    • BAM is the binary format of SAM

http://samtools.sourceforge.net/


Mandatory Alignment Fields

http://samtools.sourceforge.net/SAM1.pdf


Alignment Examples

Alignments in SAM format

CIGAR string -> 8M2I4M1D3M

http://samtools.sourceforge.net/SAM1.pdf


Annotation formats
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


GTF

Annotation data format

http://useast.ensembl.org/info/website/upload/gff.html


BED format

Annotation data format

chr1 86114265 86116346 nsv433165

chr2 1841774 1846089 nsv433166

chr16 2950446 2955264 nsv433167

chr17 14350387 14351933 nsv433168

chr17 32831694 32832761 nsv433169

chr17 32831694 32832761 nsv433170

chr18 61880550 61881930 nsv433171

chr1 16759829 16778548 chr1:21667704 270866 -

chr1 16763194 16784844 chr1:146691804 407277 +

chr1 16763194 16784844 chr1:144004664 408925 -

chr1 16763194 16779513 chr1:142857141 291416 -

chr1 16763194 16779513 chr1:143522082 293473 -

chr1 16763194 16778548 chr1:146844175 284555 -

chr1 16763194 16778548 chr1:147006260 284948 -

chr1 16763411 16784844 chr1:144747517 405362 +


BED: zero based, start inclusive, stop exclusive

Length = stop-start

GTF/GFF: one based, inclusive

Length = stop-start+1


GRCh37

NCBI36


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