Hi,

I have some metagenomic whole genome sequencing pair-end data obtained with a NovaSeq Illumina machine and a fixed length of 150bp. The sequencing has been done by a private company and the data came already trimmed and filtered but they do not pass all of the fastQC tests so I performed my self some additional cleaning steps. However, after all of this, I end up with some reads longer than 150bp and now I am confused. Is this ok?

Here what I have done, step-by-step:

Trim NEXTERA sequences with Trimmomatic

for FILE in $(ls *_R1.fastq | sed 's/_R1.fastq//'); do trimmomatic PE -phred33 ${FILE}_R1.fastq ${FILE}_R2.fastq path/${FILE}_trimmed_1.fastq path/${FILE}_unpaired_1.fastq path/${FILE}_trimmed_2.fastq path/${FILE}_unpaired_2.fastq ILLUMINACLIP:path/nextera.fa.txt:2:30:10; done  

Remove overrepresented G-polymers with bbduk

for FILE in $(ls *_trimmed_1.fastq | sed 's/_trimmed_1.fastq//'); do bbduk.sh in1=${FILE}_trimmed_1.fastq in2=${FILE}_trimmed_2.fastq out1=${FILE}_bbduk_1.fastq out2=${FILE}_bbduk_2.fastq entropy=0.5 entropywindow=50 entropyk=5; done 

Remove duplicate reads with seqkit

for FILE in $(ls *.fastq | sed 's/.fastq//'); do seqkit rmdup -s ${FILE}.fastq > ${FILE}.unique.fastq; done

Merge pair ends

for FILE in $(ls *.fastq | sed 's/.fastq//'); do pear -j 4 -n 30 -f ${FILE}_1.unique.fastq -r ${FILE}_2.unique.fastq -o ${FILE}.merged.unique.fastq; done

Any help about theory/codes/tools would be more than appreciated.

Thanks

here below the plot of the reads length