eBook: Essential Guide to Best Practices in the Lab V - 10

cules. Knowing the amount of viable DNA going into
library prep is also useful for calculating the theoretical
depth of coverage that can be obtained for each
sample. For example, if the final library contains
fewer genome equivalents than the coverage depth
required, no amount of additional library amplification
and/or sequencing will provide enough unique
reads to reach the desired coverage depth; realizing
this early on can avoid expensive re-sequencing of
suboptimal samples.
Use high-quality library prep reagents
and well-designed probes
Efficient library preparation requires a workflow that
is compatible with the type of input being used (e.g.,
FFPE, low-quality, low-input, cfDNA); has a high conversion
rate (the percentage of input DNA molecules
that successfully ligate to sequencing adapters); and
that preserves the complexity of the input sample.
Other important factors to consider, especially
in target enrichment workflows, are GC bias and
coverage uniformity. The amount of GC bias in
target-enriched sequencing data can be highly
variable, resulting in extremely low coverage of
some regions (requiring additional sequencing)
and over-sequencing of others (resulting in wasted
reads). In other cases, some target regions may
be missed entirely (target dropout); when this occurs,
the target-enriched libraries must be re-created
as additional sequencing will not provide
data for these regions. Using well-designed target
enrichment probes produced by a reliable manufacturer
can reduce these problems. Additionally,
high-quality probes can enable shorter hybridization
times, reduced workflow length, and increased
sample throughput without compromising
data quality.
10

eBook: Essential Guide to Best Practices in the Lab V

Table of Contents for the Digital Edition of eBook: Essential Guide to Best Practices in the Lab V

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eBook: Essential Guide to Best Practices in the Lab V - 1
eBook: Essential Guide to Best Practices in the Lab V - Contents
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