Quality Control and Preprocessing Talk

Contents

• Data formats

– Fasta and Fastq formats

– Sequence quality encoding

• Quality Control (QC)

– Evaluation of sequence quality

– Quality control tools

– Addressing QC with FastQC

– Typical artifacts and sequence filtering

Data formats

• Text-based formats
• If not compressed, it can be huge
• Many bioinformatics packages have parsers for these (Parse: break up into more-easily handled components).

Fastq format 1

• : separates the information field in the ID line
• 6 is the flowcell lane
• 73 is the tile number
• 941 and 1973, the x- and y- coordinates of the cluster within the file
• #0, index number for a multiplexed sample (0 for no indexing)
• /1 the first member of a pair, /2 for the second, nothing if single-ended.

Data format 2

• Fourth line is a quality indicator for each base called.
• The quality value is encoded, so that is can be single character referring to each base in sequence.
• encoding is based on the ASCII code, the command line shows the gory details: man ascii

Sequence Data Format

Raw sequence data format (Flat/Binary files)

• Fasta, Fastq, HDF5 (this latter a new complex binary format).
• Others: http://en.wikipedia.org/wiki/List_of_file_formats#Biology

Processed (often into annotations) sequence data format (Flat files)

• Column separated files containing genomic features and their chromosomal coordinates.
• GFF and GTF
• BED

GFF

• Column separated file format contains features located at chromosomal locations
• Not a compact format
• Several versions

– GFF 3 most currently used – GFF 2.5 is also called GTF (used at Ensembl for describing gene features)

GFF3 file example

GFF graphicalGFF

• representation
• structure

GFF3 can describes the representation of a protein-coding gene (From Sascha Steinbiss' genome-tools suite of programs: http://genometools.org/)

BED

• Created by UCSC Genome team
• Contains similar information to the GFF, but optimized for viewing in the UCSC genome browser

- Essentially about features and ranges.

• BIG BED, optimized for next gen data – essentially a binary version

– It can be displayed at UCSC Web browser (even several Gbs !!)

Quality Control

Evaluation of sequence quality

• Primary tool to assess sequencing
• Evaluating sequences in depth is a valuable approach to assess how reliable our results will be.
• QC determines posterior filtering

- Any filtering decision will affect downstream analysis.

• QC must be run after every critical step.

Quality control tools 1

• Fastx-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html)

• NGS QC Toolkit (http://www.nipgr.res.in/ngsqctoolkit.html)

Quality control tools

• FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/)

Multiple Sample Quality control

• Multiqc(http://multiqc.info)

- uses FastQC output

Addressing QC with FastQC

• various screen devoted to plots of the following:

- Basic stats

- Per base sequence quality

- Per read sequence quality

- Per base sequence content

- Per base GC content

- Per sequence GC content

- Per base N content

- Sequence length distribution

- Duplicate sequences

- Overrepresented sequences

- Overrepresented k-mers

Examples on web:

- Good quality:

http://www.bioinformatics.babraham.ac.uk/projects/fastqc/good_sequence_short_fastqc.html

- Bad quality:

http://www.bioinformatics.babraham.ac.uk/projects/fastqc/bad_sequence_fastqc.html

Per base sequence quality, good

Good data = Consistent high quality along the read

Per base sequence quality, bad

Bad data = Quality decreases towards the end of the read and High variance

Addressing QC with FastQC

Per sequence quality scores

Per tile sequence quality 1

Per tile sequence quality 2

Per base sequence content

Per base GC content

Per sequence GC content

Per sequence N content

Sequence duplication levels

Overrepresented sequences and k-mer content

Sequence Filtering 1

• It is important to remove bad quality data as our confidence on downstream analysis will be improved.

Sequence Filtering 2

Sequence filtering tools

• Fastq-mcf

- https://code.google.com/p/ea-utils/wiki/FastqMcf

• Cutadapt

- https://code.google.com/p/cutadapt/

• SeqTK

- https://github.com/lh3/seqtk

• Trimmomatic

- http://www.usadellab.org/cms/?page=trimmomatic)

Next

Practical sequence filtering session