Part 3: Single cell genome assembly

3.2 Pre-processing

Before assembly you can do several pre-processing steps. Here we will focus on quality trimming of the reads with the software Trimmomatic . Keep in mind which dataset you will make assemblies for, perhaps you do not need to trim your dataset.

Trimming reads with Trimmomatic

Trimmomatic is a collection of tools for reads pre-treatment. We will use it to cut the Illumina adaptor with ILLUMINACLIP giving the location of the sequences to be removed, as well as some parameters describing for example how many mismatches shold be tolerated. LEADING and TRAILING removes bases below the given threshold from the beginning and end of the read. SLIDINGWINDOW performs a sliding window quality trimming in a given window length (4) using given quality threshold (15). Finally, MINLEN specifies a length threshold for reads to be kept. In case after all of the pre-processing steps the reads get shorter than this threshold (36) they are discarded. You can read up on the details of these commands on the Trimmomatic website

We first create a folder to store the trimmed files:

mkdir trimmed

Make sure before you run this command that ‘trim’ variable is set correctly.

java -jar $TRIMMOMATIC_HOME/trimmomatic.jar PE -phred33 \
-basein G5_${sample}_R1_001.fastq -baseout trimmed/G5_${sample}${trim}.fastq \
ILLUMINACLIP:$TRIMMOMATIC_HOME/adapters/NexteraPE-PE.fa:2:30:10 \
LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

The previous command create 4 different files in the folder called trimmed:

• G5_${sample}_Trimmomatic_1P.fastq: paired forward reads
• G5_${sample}_Trimmomatic_2P.fastq: paired reverse reads
• G5_${sample}_Trimmomatic_1U.fastq: unpaired forward reads
• G5_${sample}_Trimmomatic_2U.fastq: unpaired reverse reads

We will merge both unpaired files into a single one:

cat trimmed/G5_${sample}_Trimmomatic_1U.fastq trimmed/G5_${sample}_Trimmomatic_2U.fastq > trimmed/G5_${sample}_Trimmomatic_U.fastq
#rm trimmed/G5_${sample}_Trimmomatic_1U.fastq trimmed/G5_${sample}_Trimmomatic_2U.fastq