Illumina - practical

• example workflow with a few steps included
• part of the SLU bioinformatics course
• in general always check the --help for each program

1. Cleaning up Reads

• fastqc gives you an overview whats may be wrong with your reads

N50 is defined as the minimum contig length needed to cover 50% of the genome. It means, half of the genome sequence is in contigs larger than or equal the N50 contig size

PHRED quality score: gives the probability of an incorrect base call for each base. Score 10 = 1 error in 10 bases. Score 20 = 1 error in 100. Score 30 = 1 in 1000 and so on. It's encoded from 0 to 93 as ASCII symbols in the fastq format. scythe removes adapters and or barcodes, or remove the "overrepresented sequence" that fastqc is reporting * Download Illumina adapter files here sickle for removal of low quality sequences; -s /dev/null moves singleton files to trash * multiqc takes all fastqc reports in this directory and creates and combined html file

fastqc <reads.fastq>

scythe -a <adapters.fasta> <reads.fastq>

sickle pe -f <fileinputfwd> -r <fileinputrev> -o <outputfwd> -p <outpudrev> -t sanger -s /dev/null -q 25

fastqc <trimmed_reads.fastq>

multiqc .

2.a megahit assembly

megahit -1 fwd.fastq.gz -2 rwd.fastq.gz -o m_genitalium

2.b or spades assembly

spades.py -k 21,33,55,77 --careful --only-assembler -1 <fwd read>.fastq.gz -2 <rws reads>.fastq.gz -o spade_output -m 16

Quality control

• with quast

quast.py m_genitalium.fasta -o m_genitalium_report

3. Index creation

• with bowtie and bowtie/samtool pipe

bowtie2-build *ASSEMBLYFILE* *m_genitalium123*
bowtie2 -x *m_genitalium123* -1 ERR486840_1.fastq.gz -2 ERR486840_2.fastq.gz | samtools view -bS -o m_genitalium.bam

• -x is the foldername (created with the first bowtie2 command)

4. Sort with samtools and build a index

samtools sort m_genitalium.bam -o m_genitalium.sorted.bam
samtools index m_genitalium.sorted.bam

5. Starting pylon for missmatch correction

pilon --genome m_genitalium.fasta --frags m_genitalium.sorted.bam --output m_genitalium_improved

6. Quality control

• with busco.py
• needs a database (wget from busco homepage)
• tells you how many conserved genes could be found in the assembly (QC)

wget http://busco.ezlab.org/datasets/bacteria_odb9.tar.gz

BUSCO.py -i m_genitalium_improved.fasta -l bacteria_odb9 -o busco_genitalium -m genome

-i input = output of pylon -l is the database from wget -m genome = which type