Step 1: Quality Control (FastQC and MultiQC) - aduckwo4-boop/EPP622-2026-SNPCalling GitHub Wiki

Step 1: Quality Control (FastQC + MultiQC)

Commands

FastQC was used to assess the quality of the raw sequencing reads for all four samples. MultiQC was then used to combine the individual FastQC reports to compare sequencing quality, adapter contamination, and other potential issues across samples.

cd /lustre/isaac24/proj/UTK0505/aduckwo4/622_test2
mkdir 01_fastqc
cd 01_fastqc
ln -s ../reads/*fastq.gz
nano fastqc.bash

# ln -s: creates symbolic links to the raw FASTQ files.
# nano: opens a text editor to create the FastQC script.

Created fastqc.bash

for file in *fastq.gz
do
    fastqc -t 2 $file
done

# for: loops through each Fastq file in the directory.
# fastqc: analyzes sequencing read quality.
# -t 2: uses two threads to process the reads.
# $file: specifies the current Fastq file being analyzed.
# done: ends the for loop.

Ran FastQC and MultiQC

bash fastqc.bash
multiqc . 
scp [email protected]:/lustre/isaac24/proj/UTK0505/aduckwo4/622_test2/01_fastqc/multiqc_report.html ~/Downloads

# multiqc .: combines FastQC results into a single report
# scp: securely copies the MultiQC report

MultiQC gives a rounded number for total reads and does not report total bases per sample. To find out those numbers use this:

for f in *.fastq.gz; do
  echo -ne "$f\t"
  zcat "$f" | awk 'NR%4==2{r++; b+=length($0)} END{printf "%d\t%d\t%.1f\n", r, b, b/r}'
done | column -t

This gives the total reads, then the total bases, then the average read length for each file.

Results

MultiQC report path and name: /lustre/isaac24/proj/UTK0505/aduckwo4/622_test2/01_fastqc/multiqc_report.html

One sample has adapter and polyG issues, one sample has low quality, one sample has low coverage, and one is problem free. Label each sample below with its quality issues. For each sample, report R1 + R2.

Sample	Total reads	Total bases	Quality issues?
A	R1:804650 R2:804650	R1:120697500 R2:120697500	Problem Free
B	R1:160930 R2:160930	R1:24139500 R2:24139500	Low Coverage
C	R1:804650 R2:804650	R1:120697500 R2:120697500	Adapter Contamination and polyG issues
D	R1:804650 R2:804650	R1:120697500 R2:120697500	Low Quality