Sequencing Data Quality Saulo Aflitos

Read (≈100bp) Contig (≈2Kbp) Scaffold (≈ 2Mbp) Pseudo Molecule (Super Scaffold) Paired-End Mate-Pair LowComplexityRegion Assembly - Concepts

Scaffold (≈ 2Mbp) Paired-End Mate-Pair LowComplexityRegion Pseudo Molecule (Super Scaffold) Scaffolding

Assembly

Repeats?! Scaffolding

Goldberg SMD et al x 3x2x 3x 1x Consensus Reads Contig Depth of Coverage Reality

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA NAAACGTACGTAAAANAAACGTACGTAAAA A/C A C 95% ±550% ±10 Heterozygozity

Raw Filtered Consequences of Data Cleaning

Sequencing Shotgun RNAseq

Sequencing Paired End Mate Pair

Shred Size Selection Adapter Sequencing Genome Ultrasound Physical RE Gel Beads ID Binding to Surface Circularization Illumina 454 PacBio Sample Preparation

Shredding

Size Selection

100bp Insert Size 150bp-2Kbp Illumina PE Read Length Sequencing

Insert Size 2K-20Kbp Read Length 500bp 454 MP 150bp Sequencing

Data

Machine Name Read ID (unique) Encoded Quality 0-40 Chance of being wrong FastQ

FastQ Format

% FastQ Statistics

Cleaning

Sequence duplication Per base N-content Per base GC content Per base sequence quality Per sequence quality Sequence length distribution Per base sequence content Contamination screen fastq screen Per sequence GC content FastQC Quality Checking Tool

SolexaQA Cleaning Tool

Exercise Create “cleaning” folder – mkdir cleaning; cd cleaning Inside it, run: wget -O saulo.bash Run it with: bash saulo.bash This will download FastQC and SolexaQA – FASTQC HELP : – FASTQC TUTORIAL: – FASTQC MANUAL : – SolexaQA Help : Run FastQC:./FastQC/fastqc & File > open [Files of Type = FastQ files]

Exercise Verify the two.fq files (you can use less ): – bad_MiSeq_dataset.fq – good_MiSeq_dataset.fq Clean the bad dataset with SolexaQA’s DynamicTrim.pl script: – perl SolexaQA_v.2.1/DynamicTrim.pl ► bad_MiSeq_dataset.fq -h 25 Verify the improvement (or not) by opening – bad_MiSeq_dataset.fq.trimmed

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