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Amplicon read layouts

See also
UPARSE pipeline
Read quality filtering
FASTQ format options
  Quality scores

  Global trimming

Illumina paired read with overlap
The reads contain biological sequence only. The pair can be merged by fastq_mergepairs to obtain a consensus biological sequence.

Illumina paired reads with staggered overlap
When the alignment is staggered, one or both reads extend into non-biological sequence. The pair can be merged by fastq_mergepairs to obtain a consensus biological sequence. The non-biological sequence will be deleted automatically because fastq_mergepairs detects staggered alignments and deletes terminal gaps before building a consensus sequence.

Illumina paired reads with no overlap
The reads contain biological sequence only. The reads can be combined using fastq_join which inserts a spacer (default 8 Ns) between the reads. OTUs can be constructed from joined reads by dereplicating (derep_fulllength command) and clustering with cluster_otus. This is valid even if some of the reads overlap, giving you an option for processing paired reads where varying amplicon length means that you sometimes get an overlap but not always, as it typically does with ITS. For analysis that can't deal with joined sequences you can trim to the end of the first read using fastx_truncate. Taxonomy prediction with utax works fine with joined sequences because any duplicated sequence in an overlap segment will only count once to the unique words in the sequence.

Illumina unpaired read
An unpaired read may extend into non-biological sequence at the end of the sequencing construct. This will happen if the read length is longer than some of your amplicons. To remove non-biological sequence, you can use the search_oligodb command to find the reverse primer. You will have to write your own script to trim to the primer local as usearch currently does not have a command for this.

454 read
A typical 454 read starts with a control sequence (usually TCAG), followed by the barcode and forward primer. Sometimes the read extends through the reverse primer. I provide a python script fastq_strip_barcode_relabel.py to extract the biological sequence from this type of read. This script does not find or remove the reverse primer, but that usually doesn't matter because the reads will be trimmed to a fixed length before clustering which should delete any reverse primer sequences with 16S at least (with ITS it may be more complicated due to the greater variation in amplicon length).

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