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Old 05-09-2019, 01:36 PM   #4
kmcarr
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Location: USA, Midwest

Join Date: May 2008
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Quote:
Originally Posted by SDPA_Pet View Post
Thanks. Do you have any idea about the so-called sequencing primers?
The custom sequencing and index primers are complementary to the target specific portions of the original PCR primers, plus the pad and linker. For example, if the amplicon you are sequencing is 16S-V4 generated with the usual 515F forward primer then the custom R1 sequencing primer would be:

<pad><link><515F>

or

TATGGTAATT GT GTGCCAGCMGCCGCGGTAA

These are all described in the WetLab SOP, Appendix C referred to in my post above. (Look for "Generic read 1 primer design", etc.)

The reason custom sequencing primers complementary to the 3' ends of your PCR primers are used is so that you aren't wasting sequencing data reading through your PCR primers which you would then need to trim off the 5' ends of your reads. This method was also used in the protocol developed in the Knight lab for their 16S MiSeq libraries. Figure 1 in the reference below gives a good presentation of the positions of these sequencing and index primers and their relationship to the ends of the PCR primers.

Caporaso, J. G. et al. 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, 45164522 (2011).
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