...
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 barcoesbarcodes:
Uri Laserson_6957574_6123588.XLSThe forward barcodes are present here:
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.
Name | Sequence |
PE-IV-PCR-097 | CATTTCGCT |
PE-IV-PCR-098 | TTGCTCGTG |
PE-IV-PCR-099 | TCCGCTCAC |
PE-IV-PCR-100 | CCCAACAAA |
PE-IV-PCR-101 | GCAGACCAA |
PE-IV-PCR-102 | TGGCGATAT |
PE-IV-PCR-103 | TGGTTCTGC |
PE-IV-PCR-104 | GGTACGAGT |
PE-IV-PCR-105 | ACCCGTTCG |