Data from: Indexed PCR primers induce template-specific bias in large-scale DNA sequencing studies

Massively parallel sequencing is rapidly emerging as an efficient way to
quantify biodiversity at all levels, from genetic variation and expression
to ecological community assemblage. However, the number of reads produced
per sequencing run far exceeds the number required per sample for many
applications, compelling researchers to sequence multiple samples per run
in order to maximize efficiency. For studies that include a PCR step, this
can be accomplished using primers that include an index sequence allowing
sample origin to be determined after sequencing. The use of indexed
primers assumes they behave no differently than standard primers; however,
we found that indexed primers cause substantial template sequence-specific
bias, resulting in radically different profiles of the same environmental
sample. Likely the outcome of differential amplification efficiency due to
primer-template mismatch, two indexed primer sets spuriously change the
inferred sequence abundance from the same DNA extraction by up to 77.1%.
We demonstrate that a double PCR approach alleviates these effects in
applications where indexed primers are necessary.