DNA Sequencing and the Evolution of the “-Omics” - Insect Molecular Genetics - page 271

genome in a single day for the long-sought-after price of $1000” ( Ghose 2012 ). It
is, however, not able to accurately sequence long stretches of the repetitive DNA.
Pacific Biosciences released the Single Molecule Real Time (SMRT) sequencer
in April 2011. It relies on fluorescently labeled nucleotides, but it sequences just
one molecule at a time by trapping a single DNA molecule, along with a DNA
polymerase, in one of 150,000 tiny holes, and then floods the surface with all
four fluorophore-labeled nucleotides. As DNA polymerase attaches each nucleo-
tide to the sequence, laser beams of two different wavelengths illuminate each
hole, and a camera can detect the particular nucleotide being incorporated into
each chain. This machine can generate long sequencing reads ( > 1000 bp and up
to 10,000 bp), but it is expensive at US$700,000 and has a fairly high error rate
compared with the Illumina platform.
Another approach to third-generation sequencing, nanopore sequencing,
involves reading DNA sequences by resolving changes in ionic current that cor-
respond to a known DNA sequence by passing a single DNA molecule through a
protein pore of known size at a specific rate so that the DNA base can be iden-
tified accurately ( Derrington et al. 2010, Cherf et al. 2012, Manrao et al. 2012 ).
Podolak (2010) noted that third-generation sequencing methods could “change
the landscape yet again,” and the National Institutes of Health (NIH) is sup-
porting the race to achieve an effective third-generation technology with their
US$1000 human genome ( Podolak 2010 ).
7.12 Bioinformatics
As the GASP project for Drosophila indicated, it is not always easy to find genes
hidden amongst the thousands of nucleotide sequences produced by a genome-
sequencing project. Drosophila analysis methods averaged a 70% accuracy rate
in predicting structural and functional features ( Bork 2000 ). Part of the problem
is defining a “gene” ( Attwood 2000 ). Is a gene a heritable unit corresponding
to an observable phenotype? Is it genetic information that encodes a protein
or proteins? Is it the genetic information that encodes RNA? Must the gene be
translated? Is DNA a gene if the DNA is transcribed but not expressed? There
are multiple definitions of a gene; hence, the estimates of the total number of
genes in a sequenced genome can vary.
A variety of approaches have been taken to improve the process of finding
genes in eukaryotes ( Stormo 2000 ). For example, the Drosophila genome has
isochores , long > 300-kb DNA segments that are compositionally homogeneous
on the basis of GC frequencies. As is found in humans, Drosophila isochores