Which hypervariable regions do the amplicon sequences belong to: V3–V4 or V4–V5?

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Tags: processing, pipeline

#Questions about V3–V4 primers for 16S rRNA amplicon sequencing, and calculating overlap
#Comparison of Two 16S rRNA Primers (V3–V4 and V4–V5) for Studies of Arctic Microbial Communities

#Two different hypervariable regions of the bacterial 16S rRNA gene were amplified using aliquots of the isolated DNA from each sample.
- The V3–V4 region was amplified using the S-D-Bact-0341-b-S-17 (5′-CCTACGGGNGGCWGCAG-3′) and the S-D-Bact-0785-a-A-21 (5′-GACTACHVGGGTATCTAATCC-3′) primers (Klindworth et al., 2013). 
- The V4–V5 regions was amplified using the 515F-Y (5′-GTGYCAGCMGCCGCGGTAA-3′) and the 926R (5′-CCGYCAATTYMTTTRAGTTT-3′) primers (Parada et al., 2016).

A. Klindworth, E. Pruesse, T. Schweer, J. Peplies, C. Quast, M. Horn, F.O. Glöckner
Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies
Nucleic Acids Res., 41 (2013), 10.1093/nar/gks808

# check if the data are from V3-V4 or V4-V5?

mkdir pandaseq.out_V3_V4
for file in cutadapted_16S/filtered_R1/*_R1.fastq.gz; do echo "pandaseq -f ${file} -r ${file/_R1/_R2} -l 300 -p CCTACGGGNGGCWGCAG -q GACTACHVGGGTATCTAATCC  -w pandaseq.out_V3_V4/$(echo $file | cut -d'/' -f3 | cut -d'_' -f1)_merged.fasta >> LOG_pandaseq_V3_V4"; done

mkdir pandaseq.out_V4_V5
for file in cutadapted_16S/filtered_R1/*_R1.fastq.gz; do echo "pandaseq -f ${file} -r ${file/_R1/_R2} -l 300 -p GTGYCAGCMGCCGCGGTAA -q GACTACHVGGGTATCTAATCC  -w pandaseq.out_V4_V5/$(echo $file | cut -d'/' -f3 | cut -d'_' -f1)_merged.fasta >> LOG_pandaseq_V4_V5"; done

#repalce the second _R1 to _R2
pandaseq -f cutadapted_16S/filtered_R1/AH-LH_R1.fastq.gz -r cutadapted_16S/filtered_R2/AH-LH_R2.fastq.gz -l 300 -p CCTACGGGNGGCWGCAG -q GACTACHVGGGTATCTAATCC  -w pandaseq.out_V3_V4/AH-LH_merged.fasta >> LOG_pandaseq_V3_V4
pandaseq -f cutadapted_16S/filtered_R1/AH-NLH_R1.fastq.gz -r cutadapted_16S/filtered_R2/AH-NLH_R2.fastq.gz -l 300 -p CCTACGGGNGGCWGCAG -q GACTACHVGGGTATCTAATCC  -w pandaseq.out_V3_V4/AH-NLH_merged.fasta >> LOG_pandaseq_V3_V4
pandaseq -f cutadapted_16S/filtered_R1/AH-Nose_R1.fastq.gz -r cutadapted_16S/filtered_R2/AH-Nose_R2.fastq.gz -l 300 -p CCTACGGGNGGCWGCAG -q GACTACHVGGGTATCTAATCC  -w pandaseq.out_V3_V4/AH-Nose_merged.fasta >> LOG_pandaseq_V3_V4
pandaseq -f cutadapted_16S/filtered_R1/AL-LH_R1.fastq.gz -r cutadapted_16S/filtered_R2/AL-LH_R2.fastq.gz -l 300 -p CCTACGGGNGGCWGCAG -q GACTACHVGGGTATCTAATCC  -w pandaseq.out_V3_V4/AL-LH_merged.fasta >> LOG_pandaseq_V3_V4

pandaseq -f cutadapted_16S/filtered_R1/AH-LH_R1.fastq.gz -r cutadapted_16S/filtered_R2/AH-LH_R2.fastq.gz -l 300 -p GTGYCAGCMGCCGCGGTAA -q GACTACHVGGGTATCTAATCC  -w pandaseq.out_V4_V5/AH-LH_merged.fasta >> LOG_pandaseq_V4_V5
pandaseq -f cutadapted_16S/filtered_R1/AH-NLH_R1.fastq.gz -r cutadapted_16S/filtered_R2/AH-NLH_R2.fastq.gz -l 300 -p GTGYCAGCMGCCGCGGTAA -q GACTACHVGGGTATCTAATCC  -w pandaseq.out_V4_V5/AH-NLH_merged.fasta >> LOG_pandaseq_V4_V5
pandaseq -f cutadapted_16S/filtered_R1/AH-Nose_R1.fastq.gz -r cutadapted_16S/filtered_R2/AH-Nose_R2.fastq.gz -l 300 -p GTGYCAGCMGCCGCGGTAA -q GACTACHVGGGTATCTAATCC  -w pandaseq.out_V4_V5/AH-Nose_merged.fasta >> LOG_pandaseq_V4_V5
pandaseq -f cutadapted_16S/filtered_R1/AL-LH_R1.fastq.gz -r cutadapted_16S/filtered_R2/AL-LH_R2.fastq.gz -l 300 -p GTGYCAGCMGCCGCGGTAA -q GACTACHVGGGTATCTAATCC  -w pandaseq.out_V4_V5/AL-LH_merged.fasta >> LOG_pandaseq_V4_V5

#-rwxrwxrwx 1 jhuang jhuang 39590797 Jan 11 17:09 AH-LH_merged.fasta (80630 sequences)
pandaseq -f cutadapted_16S/filtered_R1/AH-LH_R1.fastq.gz -r cutadapted_16S/filtered_R2/AH-LH_R2.fastq.gz -l 300   -w pandaseq.out_notfiltering_with_primer/AH-LH_merged.fasta >> LOG_pandaseq_notfiltering_with_primer

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