eBook: Essential Guide to Best Practices in the Lab V - 50

sign, carefully designed amplicon-based TE can result
in a high on-target sequencing read percentage.
Hybridization-based TE
Hybridization-based (or capture-based) TE utilizes
single-stranded DNA or RNA oligonucleotides
(typically free in solution) as baits to capture homologous
ROIs. The double-stranded complexes
are then immobilized and washed to remove unbound,
nonspecific molecules. Then, the bound
DNA, which represents only the targeted ROIs, is
released from the baits.
Some protocols first create a sequencing library from
randomly sheared denatured genomic DNA prior to
capturing the target library molecules. Others begin
by first capturing the genomic DNA and then creating
a library from the captured sequences.
Among the advantages of hybridization-based
TE is that it is highly sensitive, so it's less likely to
miss mutations, which is particularly beneficial for
variant calling. It performs well with respect to sequencing
complexity, and offers good uniformity of
coverage. It can cover a wide expanse of genomic
regions in a single experiment. And it can be used
to capture a nearly unlimited number of targets
simply by adding more probes.
Compare and contrast: What to consider
The popularity of whole exome sequencing
(WES)-targeting the roughly 1.5% of the genome
that codes for proteins, thus vastly reducing the
amount of genetic material to be sequenced-is
a testament to the power of target enrichment.
But because many sequences remain that may
be irrelevant to the question at hand, oftentimes
even WES leads to far more sequencing, and (given
a finite amount of resources) consequently far
shallower sequencing, than is needed. While both
amplicon-based and hybridization-based TE will
significantly reduce the sequencing burden and allow
resources to be concentrated on ROIs, there are
several factors to consider.
How much time and energy do you plan to invest?
What about budget? How much sample is available
to use? Compared to amplicon-based enrichment
protocols, hybridization-based approaches require
more sample, more time, more steps, and may be
more expensive per-sample to run as well.
Yet applications like genotyping and detection of
rare variants necessitate greater sensitivity. Hybridization
captured libraries can detect mutations
present at levels down to about 1%, compared to
about 5% for amplification-based TE libraries' sensitivity.
When the probes are well-designed and of
high quality, hybridization libraries are also more
uniform, with PCR-based amplicon libraries suffering
more from PCR bias.
Sensitivity comes at the cost of specificity, though.
Amplicon-based enrichment is able to deliver a
higher percentage of on-target reads.
There is also a limit to how many targets can be
multiplexed in an amplicon-based enrichment.
That number is scalable and virtually unlimited for
a hybridization -based protocol, making it well suited
for larger probe panels.
More information can be found here.
Additional Resource
KAPA Target Enrichment Custom Panels
50

https://sequencing.roche.com/en-us/products-solutions/by-category/target-enrichment.html https://sequencing.roche.com/en-us/products-solutions/by-category/target-enrichment/hybridization/kapa-target-enrichment/kapa-te-custom-panels.html?Details=8.5x11_08-01&utm_campaign=KAPA-TE-HyperChoice&utm_source=Biocompare&utm_medium=Bench-Tip-ebook&utm_content=learn-more

eBook: Essential Guide to Best Practices in the Lab V

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