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assembly_commands.md

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Assembly Commands

This document outlines the commands used during the assembly exercise.

Read Mapping

In this section we will use bwa and minimap2 to map reads to a couple reference genomes. Then we will evaluate the read mapping.

Mapping with BWA

  • First we have to index the reference sequence
    staphb-tk bwa index <reference_sequence>

  • Then we can map the reads to the reference sequence
    staphb-tk bwa mem <reference_sequence> <read1> <read2> -o genome_mapping.sam

  • Once the reads are mapped we need to convert it to a bam to look at the mapping statistics
    staphb-tk samtools view -b genome_mapping.sam -o genome_mapping.bam

  • Then we need to sort the bam file
    staphb-tk samtools sort genome_mapping.bam -o genome_mapping_sorted.bam

Mapping Quality

  • Let's first get the average depth
    staphb-tk samtools depth genome_mapping_sorted.bam | awk '{sum+=$3} END { print "Average = ",sum/NR}'

  • Now let's get some more information about the read mapping
    staphb-tk samtools flagstats genome_mapping_sorted.bam

Assembling with Spades

To see the effects of kmer size on genome assembly we will assemble the raw reads using Spades while specifying the kmer size. We do this by supplying the kmer size using the -k flag.

17-mer Assembly

staphb-tk spades -k 17 -1 <read1> -2 <read2> -o k17_assembly

99-mer Assembly

staphb-tk spades -k 99 -1 <read1> -2 <read2> -o k99_assembly

Normal Assembly

staphb-tk spades -1 <read1> -2 <read2> -o spades_assembly

Checking Assembly Quality

To generate the assembly quality statistics you can use the program QUAST with the following command:
staphb-tk quast <assembly.fasta> -o quast_results

You can then view the results in the terminal using the following command:
cat quast_results/report.txt