Barcoded Reverse Primers for Illumina Libraries

Overview

We have designed barcodes to multiplex samples together in a single Illumina lane. Currently, only three reads supported by illumina, a forward read, a reverse read and a barcode read. However, we had incorporated an additional barcode read into the first read as well. The current design outlined in Fig S1.pdf.

Designing Illumina amplicon libraries

Any PCR amplicon (16S, TCR-beta, etc.) can be used with this scheme, since it was designed to be modular. The first step primers must contain the following:

1.) The genomic DNA primer binding sites (to attach and extend the PCR product)

2.) The forward primer must contain some site diversity. This diversity is important for cluster identification and having the first read begin with conserved primer sequence will severely impact the quality of the data. In Fig_S1.pdf, this diversity region is a string of YRYR (N's can not be used -with IDT anyway- unless specifying an equal ratio of the four bases and that might be costly). However, you can additionally add another set of barcodes and order different step one primers with the forward barcodes attached. The only caveat with this method is that you need at least four different forward barcodes in one lane to get enough diversity. The barcodes should be relatively evenly added to the sample in a ratio of 1:1:1:1 of each barcode. More than four barcodes in the forward read should increase the quality of the calls.

Specs

Here are specs for the most recent reverse barcodes:

Uri Laserson_6957574_6123588.XLS

In addition, there are 9 additional barcodes outside of the 96 in the plate above: 097-105. These can be used for multiplexing mock or control samples into your lane separately.

16S Library Preparation (Manual)