Briefly: Bioinformatics

File Formats
J Fass | 26 March 2018
 Overview
● ASCII Text
○ Sequence
■ Fasta, Fastq
○ ~Annotation
■ TSV, CSV, BED, GFF, GTF, VCF, SAM
● Binary (Data, Compressed, Executable)
○ Data
■ HDF5
■ BAM / CRAM
■ 2bit
○ Compressed
■ gzip, bzip2, bgzip
○ Executable

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 TEXT

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 Fasta
>m54050R1_180210_051102/4194473/0_1421
CCCGGCTGCCCGCCCCGCTCGAAGCGATGACTTGCCGGCGGCCCGACGCGATTAGCTGCCGCGCATGCGATGCGGCCGCGGGCGGCGTGCTGACCTGGCTGGCGGTG
TTGAGCTGCTATACATCCGGCAACAACGCTGCCCAACGACTGACCTGACCGGCCGCCTCGATCCTGGCGGCCGCCGGCCTGGCCTGCGCTTTTCCTTCTTCTCTTTC
CTTC
>m54050R1_180210_051102/4194473/1497_4602
GGCGCGCTGATCGGCAAAACGGCTGGGGCGGCCGGAACACCTTTCAACCGTCGCCAACCGCGATCGCCGCGCGCACCCCGCCTTCCGCGCCGCTGTGGCGTTCCTCG
CCCGTCCTACTCTACTGGCATCCGTCTCATTTCTCCCGCTCTTCCCTCCACCCTTTCCCTGCTCACCGCTTCCGTCTTTTTGTCAACCTCTCCTCTGGGCCGACGAC
GTCGCCGCCTACTGCGACAAAAACCGAGGTCGACAAGGCCCGCCGTTACGACCGTCACCCCGAATTCCATCCGGCTGCTCCGCGGT
>m54050R1_180210_051102/4194551/0_17688
ACCGGACGTACCGCGGGCGGGGGCCTCCCCCCCGGGTGGCTCGGGTGCAGCGCAAATCCTTTCTTTGCTGACCCACCTGCGCAGCGAGTGTGAATCTGTGCGGATCG
AGAAAACAAGAAACCCGGCGGGCCCTGCCTGACGCGCGCCCGTCCCGCCGCGCCCCTTCCGCTTGGCGACGTCGAGTTTTTGACGGGAGGTTTGTCGCTTCGACAGA
CGGGTCCGCCAGCACCCCCTCGTCGCAGTCCCGTTAACTCAGGAAGAACTCCCAGTTGGCCCGGGCATCTGCCAACGCCTCCGGGG
>m54050R1_180210_051102/4194551/17752_17812
AAACATATTATTTTTTATTACTCAAATAATTATTATATTCACCTAATTTTCTTTATTATT
>m54050R1_180210_051102/4194552/0_89
CAGATCGGGGCCCAGCATGGCCACCCGTCCTGCACGTCTACGCGCACTTCGCCGGTGGGGATCGGCAGCGGGAACGGCTCGCGGGCTGG
>m54050R1_180210_051102/4194552/162_490
GCCGCACCCGAGCCGTTCCCGCTGCCGATCCACACCGTCGACGTGCGCGTCGACGTGCAGCCGGCGTCCATGCTTGCCCCGATCTTGGGCTAACAAGCCGCTGCTGA
CACCGACGGACGCCACCGCCCGCGACCAGCTGGCCCGGGCCTCGGTGATGGCGCTGTCCTACCGTCGCGCATTCCCGCGCTCGGCATCTATCAGCCTCGGTGCCGCA
GCGTCATCGACGATGGCGAAACCGTCACTGCACGTTTTCATGACGCGGGGCAGGCAGCGAACCGGGCACATCGGGCATCTACGCCT

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 Fasta
>m54050R1_180210_051102/4194473/0_1421
CCCGGCTGCCCGCCCCGCTCGAAGCGATGACTTGCCGGCGGCCCGACGCGATTAGCTGCCGCGCATGCGATGCGGCCGCGGGCGGCGTGCTGACCTGGCTGGCGGTG
TTGAGCTGCTATACATCCGGCAACAACGCTGCCCAACGACTGACCTGACCGGCCGCCTCGATCCTGGCGGCCGCCGGCCTGGCCTGCGCTTTTCCTTCTTCTCTTTC
CTTC

Header symbol “>” also redirects stuff into files, so be careful using > in bash commands!

Header text (sequence ID) has formats particular to different organizations and different software, but
really has no consistent rules that you can rely on.

Sequence can contain: newline characters (“\n”), ACGT, N, acgt, n, x, . or - (gaps), IUPAC ambiguity
codes BDHV etc., alternates like [A/T], amino acid single letter codes (protein fasta; sometimes file
name is ‘sequence.fna’ for fasta nucleic acid, or ‘sequence.faa’ for fasta amino acid)

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 Fastq … “fasta + qualities”
@SN638:981:HK7HWBCXX:2:1101:14799:2762 1:N:0:TTAGGC @Header1
TGGCGCAACTGCCGATCACCATCGACACCAACGGGTATCTGGTCGCCAAC
+ Sequence
GGGGGIIIIIIIIIIIIIIIGIIIIIIIIIIIIIIIIIIIIIIIIIIIIG +Header2
@SN638:981:HK7HWBCXX:2:1101:14784:2782 1:N:0:TTAGGC
CATCATCGAGGACAGCGCCGGTGACCTGGCGGCCCGCATCGGTGCCCCCC
Qualities
+
GGGGGIIIIIIIIIIIIIIIIIIIIIIIIIGIIIIIIIIIIIIIIGIIII
@SN638:981:HK7HWBCXX:2:1101:14983:2799 1:N:0:TTAGGC
Blocks of four lines for each sequence (sequences
CGGCGCCGTTGCTGCTGCTGCCGGTGCTGCTTTCGGCGCTGATCGTGCGG shouldn’t occupy more than one line, as they can in
+
fasta). Second header line (starting with “+”) is
GGGGGIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGIII
@SN638:981:HK7HWBCXX:2:1101:14763:2901 1:N:0:TTAGGC mandatory, sometimes contains the same header as
CCTGACGACGGCACGAAGGACCTCTTCGTCCACTACTCCGAGATCCAGGG the first line (that starts with “@”). Why??
+
GAGGGIGIGGGGGGGGIA.<GGGIGGAGGGGIIGIIGGIIIG<GA.<<GA
The nth quality character applies to the nth nucleotide,
and is a number that is encoded in a single character
from the ASCII table.

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 Fastq … “fasta + qualities”
@SN638:981:HK7HWBCXX:2:1101:14799:2762 1:N:0:TTAGGC The “I” for base 16 (“C”) means that that base has a
TGGCGCAACTGCCGATCACCATCGACACCAACGGGTATCTGGTCGCCAAC
+ quality of (I’s decimal value: 73) - 33 = 40
GGGGGIIIIIIIIIIIIIIIGIIIIIIIIIIIIIIIIIIIIIIIIIIIIG (sometimes referred to as “Q40”). Why 33? Because
@SN638:981:HK7HWBCXX:2:1101:14784:2782 1:N:0:TTAGGC
CATCATCGAGGACAGCGCCGGTGACCTGGCGGCCCGCATCGGTGCCCCCC
there are 32 non-printable “characters” at the
+ beginning of the ASCII table! (type ‘man ascii’)
GGGGGIIIIIIIIIIIIIIIIIIIIIIIIIGIIIIIIIIIIIIIIGIIII
@SN638:981:HK7HWBCXX:2:1101:14983:2799 1:N:0:TTAGGC
CGGCGCCGTTGCTGCTGCTGCCGGTGCTGCTTTCGGCGCTGATCGTGCGG Q40 means that the probability of error (that C is
+
actually the wrong basecall) is:
GGGGGIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGIII
@SN638:981:HK7HWBCXX:2:1101:14763:2901 1:N:0:TTAGGC
CCTGACGACGGCACGAAGGACCTCTTCGTCCACTACTCCGAGATCCAGGG pe = 10(-40 ／ 10) = 0.0001, or 1 in 10,000
+
GAGGGIGIGGGGGGGGIA.<GGGIGGAGGGGIIGIIGGIIIG<GA.<<GA

see also: https://en.wikipedia.org/wiki/FASTQ_format

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 CSV and TSV - comma/tab-separated values
B01 B02 B03 B04 For example, abundances of mRNAs from genes (count data).
PDCD1 0 0 0 0
GAL3ST2 0 0 0 0
D2HGDH 55 71 89 101 (First tab character - “\t” - in column names sometimes omitted for ease of
ING5 1 1 1 1
DTYMK 2 5 7 12
reading by R scripts).
ATG4B 0 0 0 0
THAP4 136 158 85 161
BOK 0 0 0 0
STK25 145 175 195 141

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 BED - tsv with defined column meanings
chr7 127471196 127472363 Pos1 0 + column meaning
chr7 127472363 127473530 Pos2 0 +
chr7 127473530 127474697 Pos3 0 + 1 chromosome name
chr7 127474697 127475864 Pos4 0 + 2 feature start coordinate (0-based...?)
chr7 127475864 127477031 Neg1 0 -
chr7 127477031 127478198 Neg2 0 -
3 feature stop coordinate (0-based...?)
chr7 127478198 127479365 Neg3 0 - 4 feature name
chr7 127479365 127480532 Pos5 0 +
chr7 127480532 127481699 Neg4 0 -
5 score (1-1000)
6 strand (‘+’ or ‘-’ or ‘.’ for unknown or not applicable)
… …

Number of columns used shouldn’t vary within a particular file.

see also:
https://genome.ucsc.edu/FAQ/FAQformat.html#format1

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 GFF / GTF - tsv with defined column meanings
chr22 TeleGene enhancer 10000000 10001000 500 + . touch1
chr22 TeleGene promoter 10010000 10010100 900 + . touch1
chr22 TeleGene promoter 10020000 10025000 800 - . touch2

column meaning
1 chromosome / scaffold name
2 source (e.g. software that generated this feature / gene call)
3 feature name (e.g. “exon1”, “enhance”r, “3’-UTR”)
4 feature start coordinate (1-based)
GTF is newer, and shares the first
5 feature stop coordinate (1-based) eight (8) columns. Column 9 has
6 score (1-1000) additional restrictions in format
7 strand (‘+’ or ‘-’ or ‘.’ for unknown or not applicable) (gene_id, transcript_id, etc.)
8 reading frame (0, 1, 2, or “.” if N/A)
9 group (allows grouping features together)

see also: https://genome.ucsc.edu/FAQ/FAQformat.html#format3

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 VCF - tsv with defined column meanings
##fileformat=VCFv4.2
##fileDate=20090805
##source=myImputationProgramV3.1
##reference=file:///seq/references/1000GenomesPilot-NCBI36.fasta
##contig=<ID=20,length=62435964,assembly=B36,md5=f126cdf8a6e0c7f379d618ff66beb2da,species="Homo sapiens",taxonomy=x>
##phasing=partial
##INFO=<ID=NS,Number=1,Type=Integer,Description="Number of Samples With Data">
##INFO=<ID=DP,Number=1,Type=Integer,Description="Total Depth">
##INFO=<ID=AF,Number=A,Type=Float,Description="Allele Frequency">
##INFO=<ID=AA,Number=1,Type=String,Description="Ancestral Allele">
##INFO=<ID=DB,Number=0,Type=Flag,Description="dbSNP membership, build 129">
##INFO=<ID=H2,Number=0,Type=Flag,Description="HapMap2 membership">
##FILTER=<ID=q10,Description="Quality below 10">
##FILTER=<ID=s50,Description="Less than 50% of samples have data">
##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
##FORMAT=<ID=GQ,Number=1,Type=Integer,Description="Genotype Quality">
##FORMAT=<ID=DP,Number=1,Type=Integer,Description="Read Depth">
##FORMAT=<ID=HQ,Number=2,Type=Integer,Description="Haplotype Quality">
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT sample07 ...
20 14370 rs6054257 G A 29 PASS NS=3;DP=14;AF=0.5;DB;H2 GT:GQ:DP:HQ 0|0:48:1:51,51
20 17330 . T A 3 q10 NS=3;DP=11;AF=0.017 GT:GQ:DP:HQ 0|0:49:3:58,50
20 111069 rs6040355 A G,T 67 PASS NS=2;DP=10;AF=0.333,0.667;AA=T;DB GT:GQ:DP:HQ 1|2:21:6:23,27
20 123027 . T . 47 PASS NS=3;DP=13;AA=T GT:GQ:DP:HQ 0|0:54:7:56,60
20 123457 microsat1 GTC G,GTCT 50 PASS NS=3;DP=9;AA=G GT:GQ:DP 0/1:35:4

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 SAM - tsv with defined column meanings
http://www.htslib.org/

See also samtools man page: http://samtools.sourceforge.net/

SAM spec grew out of 1000 Genomes Project (see Li et al. 2009 Bioinformatics
25:2078)

SAM is plain text; BAM is binary, compressed version of SAM; CRAM is further
compressed but not widely used / recognizable by many tools.

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 SAM - tsv with defined column meanings
[...]
@SQ SN:ctg103993 LN:217
@SQ SN:ctg103994 LN:222
@SQ SN:ctg103995 LN:205
@SQ SN:ctg103996 LN:210
@PG ID:bwa PN:bwa VN:0.7.13-r1126 CL:bwa mem -t 4 -M ../../01_Reference/Transcriptome-Contigs-Build2.fna
../../02-Cleaned/3E/3E_SE.fastq
@PG ID:bwa-7BC92A6F PN:bwa VN:0.7.13-r1126 CL:bwa mem -t 4 -M ../../01_Reference/Transcriptome-Contigs-Build2.fna
../../02-Cleaned/3E/3E_R1.fastq ../../02-Cleaned/3E/3E_R2.fastq
K00188:264:HG3WJBBXX:1:1116:14692:35180#0 121 ctg2 128 58 101M = 128 0
AAGTCTCGACCAAGTGGTTCAGATGGTGACACAGATGTTAGCCCCATCCACCATTCAGTTGCCGTTTTGATAGCTGGAAATCCTGTAAACACAATGCTGAG
FJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA NM:i:10
K00188:264:HG3WJBBXX:1:1116:14692:35180#0 181 ctg2 128 0 * = 128 0
TTTAGTTTTAATTTTTGACTTTGAATAGCGGGAGTCCAGATCGTGTGAACACAGCAGACTGAGCACTCCATTGACAGCCTTCTTCTGTACTTTAGCTATCC
FJFJJFAAJF7F7JJJJAFFFAF<7<AFFJJJFJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJFAJJJJJJJJFFFJJJJJJJJJJJFFJJJJJJJFFFAA AS:i:0 XS:i:0
K00188:264:HG3WJBBXX:1:1202:11028:9596#0 121 ctg5 45 60 101M = 45 0
TTCTTTTTTCTACAGTTCATTGTCTGTATAAAGTATGCATCAGGAACAATCTGACTAGGAAGGTAAATAATGTAAAACAGATGATTATTGTATGAAAGTTG
JJJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA NM:i:8
K00188:264:HG3WJBBXX:1:1202:11028:9596#0 181 ctg5 45 0 * = 45 0
TCAGCTGTATTAGTAATTTAGTAGAAAAGGTCTTGAGAGAATTATGTTTTTTAAAAATCCACATCACTTCAAACAAAAAGCCCCATTAGAATGGAGGGCCA
FJFJJJJJJFJJJJJJFFJJJJFJAJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFF-JFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA AS:i:0
[...]

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 SAM - tsv with defined column meanings

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 BINARY

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 HDF5
● “Hierarchical Data Format” used across many industries
● PacBio read data no longer comes in bas.h5 / bax.h5 files (instead, you
get BAM files) … so let’s forget about HDF5!

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 BAM / CRAM - compressed SAM
● * Don’t dump binary formats to your terminal / shell …
● Indexing both BAM and CRAM allow rapid random read access to any
coordinate range, without uncompressing whole file first
● CRAM restricts sequence alphabet, so compression ratio can be greater
● CRAM does lossy compression of base qualities, also helps
compression ratio

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 2bit
● Old format used for sequence in UCSC Genome Browser
● Can only store 4 bases per position:
○ 00 = A
○ 01 = C
○ 10 = G
○ 11 = T
○ … N? Lower case acgt for soft masking? Nope ...

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26

 Questions … comments … confusion?

UC Davis Genome Center | Bioinformatics Core | J Fass Formats 2018-03-26