Guidelines for amplicon sequencing

There are a number of experimental designs that can be used to sequence amplicons using the GS Junior. The most appropriate strategy for a particular experiment will depend on parameters including amplicon length, coverage and multiplexing requirements. In general, amplicons should be in the size range 200-600bp and should have been generated using a high-fidelity polymerase.

Sequencing may be performed bidirectionally or unidirectionally. Bidirectional data provides better supporting evidence for mutation calling and reduces context-dependent sequencing errors. However, unidirectional sequencing is well suited for 16S rDNA sequencing projects and some of the commonly used analysis pipelines have been designed with this kind of data in mind.

MID barcode sequences can be used to allow amplicons from multiple distinct samples (or multiple amplicons derived from the same sample) to be combined in a single experiment. The number of amplicon pools that can be combined in the same experiment depends upon the level of coverage required.

We are happy to discuss experimental design options including multiplexing both with and without universal-tails.

The SEQanswers forum is a good resource for queries about experimental design, practical advice on using various sequencing platforms and options for data analysis.

QIIME is an open-source software package which is a good starting point for the analysis of 16S rRNA based microbial community data.