Pipelines

HCMV pipeline

alignmentStats_ReCVR.sh is a bash script written to align multiple fastq files to a reference sequence. The script expect 4 arguments.

alignmentStats_ReCVR.sh <input_dir> <ref.fa> <signature.fa> <lib_name>

Input

<input_dir> is a directory of paired fastq files. The fastq files need to be in a directory and have the extension _R1_001.fastq and _R2_001.fastq (or _R1_001.fq and _R2_001.fq).

<ref.fa> is the name of the reference fasta file (e.g., merlin.fa)

<signature.fa> is the fasta file with the signatures of different HCMV strains.

<lib_name> is the name of the reference library when building the bowtie2 index

Dependencies

cutadapt

trim_galore

bowtie2

samtools

weeSAMv1.4

gawk

SamRemoveIndels.awk - hash-bang may need to be changed depending on your gawk installation

UniqSamPE.awk - hash-bang may need to be changed depending on your gawk installation

miRNA_Search

Pipeline

  • Step 1:

Each fastq file in the folder is trimmed using trim_galore with the following settings (–paired –length 21 –quality 10 –stringency 3).

  • Step 2:

The processed reads are subsequently aligned against the reference provided using bowtie2 allowing for a maximum fragment length of 1200 (-X 1200)

  • Step 3:

The assembly statistics are generated using weeSAMv1.4. A newer version of weeSAM is available here if you wish to have more comprehensive statistics. A number of assembly statistics are also printed to the terminal.

  • Step 4:

The library diversity is also estimated, first using SamRemoveIndels.awk and then with UniqSamPE.awk. These provide additional statistics which enable the calculation of the Ratio of total to unique coverage.

The diversity of genotypes in the sample is also estimated using miRNA_Search, which used the signature motifs to determine the number of posible strains in the sample.

  • Step 5:

The final stats are printed to output.csv in the input directory.