Quality Control Preprocessing Talk

Quality control and data pre-processing

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
• Almost every programming language has a parser

Data formats

Fastq Format: Sequence quality encoding

Sequence Data Format

Raw sequence data format (Flat/Binary files)

• Fasta, Fastq, HDF5
• Others: http://en.wikipedia.org/wiki/List_of_file_formats#Biology

Processed sequence data format (Flat files)

• Column separated files containing genomic features

and their chromosomal coordinates.

• Different files
• 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-version 3
##sequence-region ctg123 1 1497228
ctg123 . gene 1000 9000 . + . ID=gene00001;Name=EDEN
ctg123 . TF_binding_site 1000 1012 . + . Parent=gene00001
ctg123 . mRNA 1050 9000 . + . ID=mRNA00001;Parent=gene00001
ctg123 . mRNA 1050 9000 . + . ID=mRNA00002;Parent=gene00001
ctg123 . mRNA 1300 9000 . + . ID=mRNA00003;Parent=gene00001
ctg123 . exon 1300 1500 . + . Parent=mRNA00003
ctg123 . exon 1050 1500 . + . Parent=mRNA00001,mRNA00002
ctg123 . exon 3000 3902 . + . Parent=mRNA00001,mRNA00003
ctg123 . exon 5000 5500 . + . Parent=mRNA00001,mRNA00002,mRNA00003
ctg123 . exon 7000 9000 . + . Parent=mRNA00001,mRNA00002,mRNA00003
ctg123 . CDS 1201 1500 . + 0 ID=cds00001;Parent=mRNA00001
ctg123 . CDS 3000 3902 . + 0 ID=cds00001;Parent=mRNA00001
ctg123 . CDS 5000 5500 . + 0 ID=cds00001;Parent=mRNA00001
ctg123 . CDS 7000 7600 . + 0 ID=cds00001;Parent=mRNA00001
ctg123 . CDS 1201 1500 . + 0 ID=cds00002;Parent=mRNA00002
ctg123 . CDS 5000 5500 . + 0 ID=cds00002;Parent=mRNA00002
ctg123 . CDS 7000 7600 . + 0 ID=cds00002;Parent=mRNA00002
ctg123 . CDS 3301 3902 . + 0 ID=cds00003;Parent=mRNA00003
ctg123 . CDS 5000 5500 . + 2 ID=cds00003;Parent=mRNA00003
ctg123 . CDS 7000 7600 . + 2 ID=cds00003;Parent=mRNA00003
ctg123 . CDS 3391 3902 . + 0 ID=cds00004;Parent=mRNA00003
ctg123 . CDS 5000 5500 . + 2 ID=cds00004;Parent=mRNA00003
ctg123 . CDS 7000 7600 . + 2 ID=cds00004;Parent=mRNA00003

Col1	Col2	Col3	Col4	Col5	Col6	Col7	Col8	Col9
"seqid"	"source"	"type"	"start"	"end"	"score"	"strand"	"phase"	"attributes"

GFF graphicalGFF

representation structure

GFF3 can describes the representation of a protein-coding gene (From Sascha Steinbiss' genomte-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

• 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/)

Quality Control

• Addressing QC with FastQC

- 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 data = Consistent high quality along the read

Addressing QC with FastQC

Per base sequence quality

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

Addressing QC with FastQC

Per sequence quality scores

Good data = most reads are high-quality sequences

Bad data = Distribution with bi-modalities

Per tile sequence quality 1

Good data = Blue all over

Bad data = Presence of hot colours

Per tile sequence quality 2

Problems in some tiles

Filtering of reads with Q30 on 90% of the read

Per base sequence content

Good data = smooth over the read

Bad data = Sequence position bias and adapter contamination

Per base GC content

Good data = smooth over the read

Bad data = Sequence position bias and adapter contamination

Per sequence GC content

Good data = Normal distribution, Distribution fits with expected, Organism dependent

Bad data = Distribution doesn’t fit with expected. Possibility of contamination

Per sequence N content

Good data

Bad data = There are peaks of Ns per base position.

Sequence duplication levels

Good data

Bad data = High number of duplicates. Indicates some kind of enrichment bias.

• Note:

- Only few sequences are used to make this judgment.

- For RNASeq, higher number of duplicated sequences are expected.

Overrepresented sequences and k-mer content

• Exact same sequences too many times
• PCR primers, Adapters, etc.

• Note:

- Sometimes this is expected

Sequence Filtering 1

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

Sequence Filtering 2

• Mean quality
• Read length after trimming
• Percentage of bases above a quality threshold
• Adapter trimming

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