Sequence Files

Sequence files come in 2 formats, FASTQ and FASTA. Raw reads will always come as fastq files, sometimes in compressed file formats. Some programs are written to be able to handle compressed files, but I find it’s easier to decompress the files to avoid any possible hang-ups. Compressed files typically have either a .gz or .zip file extension.

FASTQ

The fatq format always has 4 lines per read.

  • @Read name
  • Read sequence
  • +some line I’ve never seen used
  • Quality scores
@M04771:122:000000000-B62C2:1:1101:13253:1557 1:N:0:0
TCCTTTTTTTTTTCTCTTTTTTCTTCTTTCCTTTTCTTCCCTCTTTCTTCTTCTTTTTTTTCCTTCTTTTTTTCCCTCCCTTTTCTTCCCTTTTTTTTTTCCTTTTTTCCTTTTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTTT
+
>>>11311>@00A012BEF1AABEE22DA21DDFF2BF210B0B112DA2DF2DB111>>E01@@11@1@EE?011B0//011BBF12101111<///</011?<11<0<?1<1<-@0<=000<<--------9@---9-9--99-///;9
@M04771:122:000000000-B62C2:1:1101:16150:1745 1:N:0:0
TACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGCGGTCTGGTCAGTCAGATGTGAAAGCCCCGGGCTCAACCTGGGAATTGCATTTGATACTGCCAGACTCGAGTGTGGTAGAGGGAAGTGGAATTCCGAGT
+
AAAA?1AD?AAA1FGGGGE?0BGFF00FFGAGFFGEEGGAGHFEA/AE>EEEBEGE/E00BGDGG>1B1BFGGFFFFECEE/<ECGC1CG<F00FA1?FDGBFGG>FFFHHG1<110?F1?.CGA>1AFFFBGH?.<AGBF0DDC<G/.:C
@M04771:122:000000000-B62C2:1:1101:14494:1756 1:N:0:0
TACGGAGGGGGTTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCGCGTAGGCGGATTATTCAGTCAGAGGTGAAATCCCAGGGCTCAACCCTGGAACTGCCTTTGATACTGCTAGTCTTGAGTTCGAGAGAGGTGAGTGGAATTCCGAGT
+
A1AA11AD@1>>AAGGEGEEFFHFEC/FGFBGFFGEEGC?/GEE>/>EEEEGFEE/>E12BF2GGD1B11?GHGB>BGCC11/CCHE11G?<00?011F<FGGGH1??1FH11F11>GHHF11GGD..0.@.?CGA0.DDEEC;C:C/.:;


FASTA

The fasta format has two lines per read.

  • >Read name
  • Read sequence
>M04771:122:000000000-B62C2:1:1101:13253:1557 1:N:0:0
TCCTTTTTTTTTTCTCTTTTTTCTTCTTTCCTTTTCTTCCCTCTTTCTTCTTCTTTTTTTTCCTTCTTTTTTTCCCTCCCTTTTCTTCCCTTTTTTTTTTCCTTTTTTCCTTTTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTTT
>M04771:122:000000000-B62C2:1:1101:16150:1745 1:N:0:0
TACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGCGGTCTGG
TCAGTCAGATGTGAAAGCCCCGGGCTCAACCTGGGAATTGCATTTGATACTGCCAGACTC
GAGTGTGGTAGAGGGAAGTGGAATTCCGAGT
>M04771:122:000000000-B62C2:1:1101:14494:1756 1:N:0:0
TACGGAGGGGGTTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCGCGTAGGCGGATTATTCAGTCAGAGGTGAAATCCCAGGGCTCAACCCTGGAACTGCCTTTGATACTGCTAGTCTTGAGTTCGAGAGAGGTGAGTGGAATTCCGAGT

Though technically FASTA formay is 2 lines per sequence, you may notice that the actual sequence for the second read here is on more than 1 line. Really, programs that use FASTA files check for the ‘>’ in the read name line to denote a new sequence.. But for best practice, use 2-line format.

Reference Database

The reference database is typically in fasta format, and is used for two purposes in this pipeline. First, it is used to compare the reads to for chimera screening. Second, it is used to classify the read clusters as belonging to particular taxa. The most commonly used databases are Silva, GreenGenes, and the RDP.

>S000494589 uncultured bacterium; YRM60L1D06060904  Lineage=Root;rootrank;Bacteria;domain;"Actinobacteria";phylum;Actinobacteria;class;Acidimicrobidae;subclass;Acidimicrobiales;order;"Acidimicrobineae";suborder;Acidimicrobiaceae;family;Acidimicrobium;genus
gcggcgtgctacacatgcagtcgtacgcggtggcacaccgagtggcgaacgggtgcgtaacacgtgaggaacctaccccg
aagtgggggataacaccgggaaaccggtgctaataccgcatacgctccccggaccgcatggtccagggagcaaagcctcc
gggcgcttcgggacggcctcgcggcctatcagcttgttggtggggtaacggcccaccaaggcgacgacgggtagctggtc
tgagaggacgatcagccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaatattgcgcaat
gggcgaaagcctgacgcagcaacgccgcgtggaggacgaaggccttcgggttgtaaactcctttcagcagggacgaaact
gacggtacctgcagaagaagccccggctaactacgtgccagcagccgcggtaag


Mapping Files

These files are used to match the barcodes of multiplexed reads to their source samples. Depending on the sequencing facility there may by 2 files, a mapping file and and I file. The mapping file is used to match the barcode the sample.

#SampleID   BarcodeSequence LinkerPrimer    Description     
ATD1    TCAGCCTCAGCC    GTGTGYCAGCMGCCGCGGTAA   E_Quiroz_plate1_A1      
ATD2    TGCAGGGAACCG    GTGTGYCAGCMGCCGCGGTAA   E_Quiroz_plate1_A2      
ATD3    CAGCGCGGCTAG    GTGTGYCAGCMGCCGCGGTAA   E_Quiroz_plate1_A3      
ATD4    ATGGACCTAGCT    GTGTGYCAGCMGCCGCGGTAA   E_Quiroz_plate1_A4      
ATD5    CTAGCCCGTTCG    GTGTGYCAGCMGCCGCGGTAA   E_Quiroz_plate1_A5

The I file matches the read to the barcode.

@M04771:122:000000000-B62C2:1:1101:13253:1557 1:N:0:0
TTTTTTTTTTCT
+
1111>33B3B13
@M04771:122:000000000-B62C2:1:1101:16150:1745 1:N:0:0
TGGGTTAACACA
+
>111>>11B3B1
@M04771:122:000000000-B62C2:1:1101:14494:1756 1:N:0:0
TTTCTTTTCTTT
+
111113313B33




Schuyler Smith
Ph.D. Student - Bioinformatics and Computational Biology
Iowa State University. Ames, IA.