1. Sequence File Formats

2. Sequence File Formats Different formats for different uses
Competing formats developed in parallel
Some easy to read, some easy to write programs
Don’t have to stick to these formats, but parsers already written!
Most formats are plain text (not .bam files!)

3. Id’s versus accessions
When people first started, they were using gene names as id’s
But too few gene names, and databases require unique ids
Now have a variety of accession numbers
The simplest id is a number that you increment, as you can (almost) never run out of IDs.

4. Standard genetic code Symbol Meaning Origin G Guanine A Adenine C
Cytosine T
Thymine R
G or A
puRine Y
T or C
pYrimidine M
A or C
aMino K
G or T
Keto N
G or A or T or C
aNy

5. Standard protein codes
One
Three
Amino acid A
Ala
Alanine M
Met
Methionine C
Cys
Cysteine N
Asn
Asparagine D
Asp
Aspartic acid P
Pro
Proline E
Glu
Glutamate R
Arg
Arginine F
Phe
Phenylalanine S
Ser
Serine G
Gly
Glycine T
Thr
Threonine H
His
Histidine V
Val
Valine I
Ile
Isoleucine W
Trp
Tryptophan K
Lys
Lysine Y
Tyr
Tyrosine L
Leu
Leucine X
Xaa
Unknown

6. Fasta Simplest file format. Easy to parse, easy to use
>identifier [optional information]
ATGACTAGCATGCATCGATCGATCGACTAGCAT G
ACTGCACTACGACGACAGCAAC
>identifier2 [optional information]
ACTAGCTCAGCTAGAGAGCTACGATCAGCACTA C
atccgatagcatgacttactACGCTAGCATCAG TCATACAT

7. GenBank
More complex, includes detailed information on genes, cds, annotation etc
Human readable
Difficult to parse
Use standard parsers (bioperl, biojava, etc)

8. LOCUS NC_001418 5833 bp ss-DNA circular PHG 17-APR-2009
DEFINITION Pseudomonas phage Pf3, complete genome.
ACCESSION NC_001418
VERSION NC_ GI:
DBLINK Project:14061
KEYWORDS .
SOURCE Pseudomonas phage Pf3
ORGANISM Pseudomonas phage Pf3
Viruses; ssDNA viruses; Inoviridae; Inovirus.
FEATURES Location/Qualifiers
source
/organism="Pseudomonas phage Pf3"
/mol_type="genomic DNA"
/host="Pseudomonas aeruginosa"
/db_xref="taxon:10872"
/note="Pf3 bacteriophage DNA from P.aeruginosa infected
with plasmid RP1."
gene join( ,1..106)
/locus_tag="Pf3_1"
/db_xref="GeneID: "
CDS join( ,1..106)
/note="orf 58, part 2"
/codon_start=1
/transl_table=11
/product="hypothetical protein"
/protein_id="NP_ "
/db_xref="GI: "
/translation="MSYYVCVQLVNDVCHEWAERSDLLSLPEGSGLQIGGMLLLLSAT
AWGIQQIARLLLNR"

9. 3241 aggtcctgtt ggccttaaga tcacccaagg gcatcttgcc agatggtacc gtcattactt
3301 atgagaaaat atcctcaatg ggtaatggct ataccttcga gcttgagtcg cttatatttg
3361 cggctcttgc tcggtcttta tgcgaattac tgggcttacg accgtcagat gttacggtct
3421 atggcgatga cataatattg ccatcagacg cgtgcagtcc tctagttgaa gttttctcct
3481 atgttggttt tcgtaccaac aagaagaaaa cgttttctag tggaccgttc cgagagtcgt
3541 gcggaaagca ctactttttg ggcgttgacg tcacaccttt ctacatacgt cgccgtatag
3601 tgagtccctc cgatctcata ctggttttga accagatgta tcgttgggcc acaattgacg
3661 gcgtatggga tcctagggta tatcctgtat acaccaagta tagacgttac cttccggaaa
3721 ttctccggag gaatgtcgtg cctgatggat acggtgatgg tgccctcgtc ggatctgtct
3781 taatcagtcc tttcgcagaa aatcgcggtt gggttcggcg tgtgccgatg attatagaca
3841 agaggaaaga ccgagttcgt gacgaatatg gttcgtatct ctacgagcta tggtcgttgc
3901 agcaactcga atgtgacagt gagttcccct ttaacgggtc gctggtcgtt ggttccactg
3961 atggcactct cgcttacgca caccgagaac ggttacctac cgttatcagt gatgccgtaa
4021 gtgcgtttga catcatgtgg ataccgtgca gtagtcgtgt cctggctccc tacggggatt
4081 tccggaggca cgaaggctct atcctaaaaa tggggtagcg cctgggaggg gtgcattatg
4141 caccctaggt tagcaatact taaactaacc ttctcaaaag agagagtgaa ggctctgctt
4201 tgccctcact cctccca //
LOCUS NC_ bp ds-RNA linear PHG 23-AUG-2008
DEFINITION Pseudomonas phage phi8 segment S, complete sequence.
ACCESSION NC_003301
VERSION NC_ GI:
DBLINK Project:14731
KEYWORDS .
SOURCE Pseudomonas phage phi8
ORGANISM Pseudomonas phage phi8
Viruses; dsRNA viruses; Cystoviridae; Cystovirus.

10. GFF3 Tab separated format Easy to parse
Columns:
Contig
Source database
Feature type
Start
Stop
Score
Strand
Phase
Attributes
Tab separated format
Easy to parse
Attributes are tag/value pairs separated by “;”

11. ASN.1
Developed as computer readable form of GenBank
Not widely used

12. ASN.1 seq { id { local id 1 }, descr { title "" }, inst {
repr raw, mol aa, length 131, topology linear, {
seq-data
iupacaa "TSPASIRPPAGPSSRPAMVSSRRTRPSPPGPRRPTGRPCCSAAPRRPQA
TGGWKTCSGTCTTSTSTRHRGRSGWSARTTTAACLRASRKSMRAACSRSAGSRPNRFA
PTLMSSCITSTTGPPAWAGDRSHE"
} } ,
iupacaa "TSPASIRPPAGPSSR RPSPPGPRRPTGRPCCSAAPRRPQAT
GGWKTCSGTCTTSTSTRHRGRSGW RASRKSMRAACSRSAGSRPNRFAPTL
MSSCITSTTGPPAWAGDRSHE"
} }

13. Base calling Need to be sure which base you have identified
Depends on the technology
Each machine includes software
Phred is an historical package developed by at U. Washington
Phred scores are probability that the base is correct 13

14. Quality values Phred 10: 1 x 101 chance that the base is wrong
Phred 99: the base is correct!
Fastq scores are the score + 33 then converted to ascii text 14

15. FastQ Based on fasta format
Contains information about the quality of the sequence
Quality comes from sequencing machines!
Four lines per sequence:
Line = identifier line before the sequence
DNA sequence
Line starting + = identifier line before the quality scores
String = quality scores as ASCII + 33

16. ASCII character codes ASCII Char 33 ! 50 2 70 F 90 Z 110 n 34 " 51 371 G 91 [
111 o 35 # 52 4 72 H 92 \
112 p 36 $ 53 5 73 I 93 ]
113 q 37 % 54 6 74 J 94 ^
114 r 38
& 55 7 75 K 95 _
115 s 39 ' 56 8 76 L 96 `
116 t 40 ( 57 9 77 M 97 a
117 u 41 ) 58 : 78 N 98 b
118 v 42 * 59 ; 79 O 99 c
119 w 43 + 60
< 80 P
100 d
120 x 44 , 61 = 81 Q
101 e
121 y 45 - 62
> 82 R
102 f
122 z 46 . 63 ? 83 S
103 g
123 { 47 / 64 @ 84 T
104 h
124 | 48 65 A 85 U
105 i
125 } 49 1 66 B 86 V
106 j
126 ~

17. fastq DNA sequence @SRR014849.1 EIXKN4201CFU84 length=93
GGGGGGGGGGGGGGGGCTTTTTTTGTTTGGAACCGA
AAGGGTTTTGAATTTCAAACCCTTTTCGGTTTCCAA
CCTTCCAAAGCAATGCCAATA
+SRR EIXKN4201CFU84 length=93
Quality scores
Note: Illumina has a format of fastq that is not compatible with everyone else’s format!

18. How to convert fastq to fasta
prinseq-lite.pl -fastq input.fastq -out_format 2