Biology Reference
In-Depth Information
from both pig and human hosts, disease processes in Ascaris as well as for
the design of new interventions against ascariasis.
ADVANCED METHODOLOGIES ESTABLISHED
TO SEQUENCE, ASSEMBLE, AND ANNOTATE
THE GEN OME AND TRANSCRIPTOMES OF
A. SUUM
Here, we provide a summary of the methodologies used for the
sequencing, assembly, and annotation of the A. suum genome and tran-
scriptome. For this genome, significant technical hurdles needed to be
overcome to enable its sequencing from a limited amount of genomic
DNA from the reproductive tract from a single female worm. In general
terms, Illumina-based technology 12 was used for the sequencing of the
genome and transcriptome of A. suum . Bioinformatic data analyses were
conducted in a Unix environment or Microsoft Excel 2007 using standard
commands; scripts required to facilitate data analysis were designed
using Perl, BioPerl, Java, and Python and are available via http://
gasserlab.org/ .
Sequencing of the Genome from the DNA
from the Reproductive Tract from a Single Adult
Female of
A. suum
Although the advent of massively parallel sequencing technologies has
vastly reduced the cost of large-scale -omics research, there has been
a number of technical and analytical challenges associated with the
de novo sequencing of large eukaryotic genomes, including, for example,
that of A. suum . Irrespective of the technology chosen, 12 these platforms
achieve massively parallelized sequencing by (1) fragmenting nucleic
acids, (2) standardizing the ends of these fragments through the ligation
of adaptors of a known sequence, and (3) immobilizing each fragment,
either on beads in an oil emulsion matrix (e.g. 454 technology or SOLID)
or on a glass slide (e.g. Illumina technology), prior to generating reads of
w
600 bp (depending on the technology used). The first versions of
these platforms achieved sequencing from one end of each fragment, with
read quality decreasing significantly with increasing read length.
Improvements to the sequencing chemistry have now enhanced read
quality and length. In addition, most platforms now achieve sequencing
from both ends of each fragment (called paired-end sequencing), 13 thus
maximizing data for subsequent assembly. Therefore, it is now possible to
sequence the ends of fragments much larger than twice the achievable
read length, leaving an unsequenced gap of known size between each pair
100
e
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