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Read preparation

See also
OTU / denoising pipeline

The table below summarizes the read preparation steps that I recommend for an OTU / denoising pipeline. Follow the links for details.

Step   Description
Understand your reads   Investigate your reads, don't blindly follow a recipe
Demultiplex   Assign reads to samples using index reads or barcodes
Merge pairs   Merge paired reads to get consensus sequences and Q scores
Strip primers   Primer-binding sequence should be removed before quality filtering
Orient   If you have reads on both strands, orient before trimming and finding uniques
Strip machine sequences   Remove machine-specific sequences e.g. TCAG for 454
Length trimming   Remove low-quality tails, make sure 3' ends align
Quality filtering   Making OTUs (but not the OTU table) needs high-quality reads
Pool samples   Reads for all samples should be combined
Discard singletons   Remove low-abundance reads, which often have errors