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of reads of each fragment. This gap provides physical/spatial information
that assists in the assembly process and, in particular, in traversing long,
repetitive regions of eukaryotic genomes. The effectiveness can be
improved further through the construction of large-insert, mate-paired
libraries,
12
whereby the physical gap or insert between the paired reads
can be up to 10 kb or more. Although this mate-paired approach is a major
advance, the shearing process used to generate DNA fragments prior to
library construction becomes increasingly inefficient as the insert size
increases, thus requiring substantially more starting template for an
effective build. Because of this inefficiency, the amount of sequence
generated from a library tends to decrease with increasing insert size as
well. Therefore, a common strategy is to generate sequence data from
libraries representing a range of insert sizes using the large amounts of
data from small-insert libraries (e.g. 200, 500, and 800 bp) to achieve a high
level of genome coverage (i.e. to ensure that all regions of the genome are
sequenced to an acceptable depth of usually
30-fold). Sequence data
from large-insert libraries (2, 5, and 10 kb) are then used to bridge
repetitive or unsequenced regions and to enhance the final assembly. We
adopted this approach to sequence the nuclear genome of
Ascaris suum
using the Illumina Hi-seq platform (
Figure 11.1
).
To do this, we purified high molecular weight genomic DNA from the
reproductive tract from a single adult female of
A. suum
by sodium-
dodecyl sulfate/proteinase K digestion,
14
followed by phenol-
chloroform extraction and ethanol precipitation.
15
We specifically
selected reproductive tissues rather than muscle because, in our experi-
ence, this tissue yields a larger amount of high molecular weight DNA
(unpublished finding). Particularly for larger fragment library construc-
tion, it is critical that genomic DNA is of high quality and high molecular
weight, and available in a sufficient amount. We assessed DNA quality
and quantity using a Qubit fluorometer dsDNA HS Kit (Invitrogen),
standard agarose gel electrophoresis and a 2100 Bioanalyzer (Agilent,
USA). The genomic DNA isolated from the reproductive tissue of a single
female was sufficient to allow the construction of two short-insert libraries
representing mean fragment sizes of 170 and 500 bp, respectively.
However, it was not enough to allow the construction of large-insert
libraries (i.e. mate-paired libraries) without further processing. Although
genomic DNA from multiple individuals could be pooled, there is
potential that genetic polymorphism among individuals could complicate
or prevent an effective assembly of sequence data. Therefore, we elected to
synthesize a substantial amount of template from an individual by whole
genome amplification (WGA) using a multi-strand displacement method
(REPLI-g Midi Kit, Qiagen). Using this technique, we generated
40
m
gof
w
high molecular weight DNA from
200 ng of template, allowing the
construction of 800 bp, and 2, 5, and 10 kb mate-paired libraries. By
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