MiSeq 2x250bp run

[Francy87]

Hi there! I have a silly question about the MiSeq read length.
I'm going to run a MiSeq plate of 16s amplicons.
When they talk about 2x250bp reads, do they include the adaptor in the calculation? So, can I expect "useful" 250bp or do I need to subtract the adaptor length?
Thanks
Francesca

[microgirl123]

The 2x250 bp does not include the adapters or indices. The read is 250 bp in each direction starting with the first base of your amplicon.

[GenoMax]

As @microgirl123 points out the 250 bp will not include adapters/indices assuming inserts in your library are of sufficient length (> 250 bp) . If the inserts in your library are small this will no longer be true.

[Brian Bushnell]

It may include the amplification primer sequence, though, which is of questionable utility.

[kmcarr]

It will include the amplification primer sequence, though, which is of questionable utility.

The reads may or may not include the amplification primer sequence depending on the protocol used to amplify/sequence them. For example, two common strategies for performing 16S V4 amplicon sequencing [1,2] add custom sequencing & index read primers to the MiSeq cartridge which are complementary to the amplification primers so that the sequence reads generated start immediately after the amplification primer.

1. Kozich, J. J., Westcott, S. L., Baxter, N. T., Highlander, S. K., & Schloss, P. D. (2013). Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Applied and Environmental Microbiology, 79(17), 5112–5120. http://doi.org/10.1128/AEM.01043-13

2. Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Lozupone, C. A., Turnbaugh, P. J., et al. (2011). Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proceedings of the National Academy of Sciences, 108 Suppl 1, 4516–4522. http://doi.org/10.1073/pnas.1000080107

[Brian Bushnell]

Live and learn. Thanks for the correction!