Single end reads are the exception and often a last resort. Paired-end reads are usually worthwhile even for short amplicons. On the one hand, you can buy kits for different numbers of cycles (e.g. 500 for PE250 or 600 for PE300) or adjust the number of cycles manually. On the other hand, overlapping reads help with error correction. However, it is possible that the number of overlapping reads is rather low because the amplicon length variation was incomplete or faulty. In this case, only the qualitatively better forward read (R1) would be used. The workflow is similar to the paired-end workflow, but instead of merging the reads, we would trim the R1 reads to avoid low quality bases at the end of the reads. We do not use quality based trimming as this would result in reads of different lengths, but rather we use fixed length trimming.

Data Processing Steps - Overview

A. Quality Control, Data and Parameter Evaluation

ls -lh y_help/A_*.report

B. Filter Reads and 3'-End Trimming

less y_help/*_FFT_R1only.report

C. Forward Primer Site Trimming

less y_hels/C_*_RMP_R1only.report

D. Size Selection & Quality Filtering

less y_help/D_*_QFR_R1only.report

E. Clustering

# Clustering
less y_help/E1_*_OTU.report
# Mapping
less y_help/E2-1_*_MAP.report #  OTU
less y_help/E2-2_*_MAP.report # zOTU
# Alignments and Stats
less y_help/E3_*_TRE.report

F. Tax Predictions

less y_help/F_*_TAX.report

G. Extra - e.g. ITSx for ITS amplicons

ls -lh y_help/G_*.report