Contact: Raymond Hovey Genomics Center - SFS

Transcription

1 Bioinformatics Lunch Seminar (Summer 2014) Every other Friday at noon minutes plus discussion Informal, ask questions anytime, start discussions Content will be based on feedback Targeted at broad audience of various levels of backgrounds and education Emphasis on Genomics Center Contact: Raymond Hovey Genomics Center - SFS rhovey@uwm.edu

2 Filetypes generated by the Genomics Center Sequencers Sanger Sequencer (ABI 3730), <750bp length, Gold standard Fasta file for each sequence (.fasta.fas.fa.fna.txt.seq ) Tracefile for each Sequence (.ab1 ) Multifasta file containing all sequences of a plate in one file

3 simple text format used by almost all programs (standard) [>] header line with a [hard return] at end Sequence (no specific requirements for line length, characters, etc. Though linebreaks inside the sequence can be problematic with some softwares, better to remove them) No standard file extension FASTA Format >URO1 uro1.seq Length: 2018 November 9, :50 Type: N Check: CGCAGAAAGAGGAGGCGCTTGCCTTCAGCTTGTGGGAAATCCCGAAGATGGCCAAAGACA ACTCAACTGTTCGTTGCTTCCAGGGCCTGCTGATTTTTGGAAATGTGATTATTGGTTGTT GCGGCATTGCCCTGACTGCGGAGTGCATCTTCTTTGTATCTGACCAACACAGCCTCTACC CACTGCTTGAAGCCACCGACAACGATGACATCTATGGGGCTGCCTGGATCGGCATATTTG TGGGCATCTGCCTCTTCTGCCTGTCTGTTCTAGGCATTGTAGGCATCATGAAGTCCAGCA GGAAAATTCTTCTGGCGTATTTCATTCTGATGTTTATAGTATATGCCTTTGAAGTGGCAT CTTGTATCACAGCAGCAACACAACAAGACTTTTTCACACCCAACCTCTTCCTGAAGCAGA TGCTAGAGAGGTACCAAAACAACAGCCCTCCAAACAATGATGACCAGTGGAAAAACAATG GAGTCACCAAAACCTGGGACAGGCTCATGCTCCAGGACAATTGCTGTGGCGTAAATGGTC CATCAGACTGGCAAAAATACACATCTGCCTTCCGGACTGAGAATAATGATGCTGACTATC CCTGGCCTCGTCAATGCTGTGTTATGAACAATCTTAAAGAACCTCTCAACCTGGAGGCTT

4 FinchTV ( Free)

5 Filetypes generated by the Genomics Center Sequencers Next Generation Sequencer (Illumina MiSeq), <250bp length Fastq files for each sample (.fastq.fq.fastq.tar.gz). Depending on the sequencer configuration there can be multiple files for a sample. Paired end reads or unpaired Read pairs in same files or separate files Soon: Pacific Biosciences Sequencer Longer reads (up to 50k bps) Can detect Modifications like Methylation High throughput for whole genome projects

6 FASTQ Format Four lines per Sequence. No file extension standard,.fastq and.fq most common Line 1 starts with followed by sequence ID and optional descriptions Line 2 is the raw sequence Line 3 begins with a + and optional repeat of the identical sequence ID from Line 1. Line 4 contains the quality scores for each base in Line 2 No single standard for lines 2 and 4 (Most files are now Sanger standard. Illumina standard is still around) Sanger Standard example 1:Y:18:ATCACG GATTTGGGGTTCAAAGCAGTATCGATCAAATAGTAAATCCATTTGTTCAACTCACAGTTT +!''*((((***+))%%%++)(%%%%).1***-+*''))**55CCF>>>>>>CCCCCCC65 Illumina Standard example TTAATTGGTAAATAAATCTCCTAATAGCTTAGATNTTACCTTNNNNNNNNNNTAGTTTCTTGAGATTTGTTGGGGGAGACATTTTTG TGATTGCCTTGAT +HWI-EAS209_0006_FC706VJ:5:58:5894:21141#ATCACG/1 efcfffffcfeefffcffffffddf`feed]`]_ba_^ [YBBBBBBBBBBRTT\]][]dddd`ddd^dddadd^BBBBBBBBBBB BBBBBBBBBBBBB

7 ID information for FASTQ Format (Sanger) Sanger Standard example 1:Y:18:ATCACG GATTTGGGGTTCAAAGCAGTATCGATCAAATAGTAAATCCATTTGTTCAACTCACAGTTT +!''*((((***+))%%%++)(%%%%).1***-+*''))**55CCF>>>>>>CCCCCCC65 EAS139 the unique instrument name 136 the run id FC706VJ the flowcell id 2 flowcell lane 2104 tile number within the flowcell lane 'x'-coordinate of the cluster within the tile 'y'-coordinate of the cluster within the tile 1 the member of a pair, 1 or 2 (paired end reads) Y Y if the read is filtered, N otherwise 18 Checksum ATCACG index sequence

8 Quality Scores for FASTQ Format (Sanger, PHRED+33) Sanger Standard example 1:Y:18:ATCACG GATTTGGGGTTCAAAGCAGTATCGATCAAATAGTAAATCCATTTGTTCAACTCACAGTTT +!''*((((***+))%%%++)(%%%%).1***-+*''))**55CCF>>>>>>CCCCCCC65 PHRED quality scores Q are logarithmically related to the base-calling error probabilities P. The quality symbol is the ASCII number of the score plus 33 (for PHRED+33) scores. PHRED Quality Score Probability of incorrect base call 10 1 in 10 90% 20 1 in % 30 1 in % 40 1 in 10, % Base call accuracy 50 1 in 100, % Range of PHRED+33 quality scores in increasing order:!"#$%&'()*+,-./ :;<=>?@abcdefghijklmnopqrstuvwxyz[\]^_`abcdefghijklmnopqrstuvwxyz{ }~ Pitfall: Older Illumina reads generated prior to Casava 1.8 used PHRED+64, meaning the score values are 31 higher.

9 Basic Alignment files generated from NGS. BAM files simple format used by almost all programs (standard) binary compressed file format (not human readable).bam file extension (plus optional.bai index file) Commonly used to transfer data as it is compressed and standardized Standard output from many alignment softwares like DNAStar, Illumina/Casava, Bowtie, etc.

10 Basic Alignment files generated from NGS. SAM files Human readable version of a BAM file Not compressed (~2x size of.bam, genome alignment files can be huge).sam file extension Easier to process than.bam files by text based approaches like awk, sed, python, perl, R, etc. Easily generated from.bam files with Samtools (samtools.sourceforge.net) A standard output of only a few alignment softwares (for example BWA) Consists of a header portion containing general information (aligner software, data size, ) and a sequence alignment portion with one line per aligned read (sequence, phred-scores, start and stop nucleotides of alignment, template ID, variants, ). Both header and sequence data formats are standardized but the actual inclusion of data is optional (!).

11 QC report for NGS data generated by Genomics Center Generated by FastQC software package ( Compressed html file (can be open with most standard browsers) Contains basics statistics and Quality data for fastq files

12 QUESTIONS? COMMENTS? AND DON T FORGET TO CHECK OUT OUR NEW HOMEPAGE AT GREATLAKESGENOMICS.UWM.EDU