3.18 Resequencing
Resequencing is another technique that is poorly named as it typically does not refer
to sequencing the same DNA over again. Rather, resequencing is the quick sequencing of a genome via second- and/or third-generation techniques and alignment of the
resulting sequence reads to a reference-quality genome sequence (Lesk 2012).
Resequencing is used to quantify diversity and investigate changes in key genes.
When short-read sequences are aligned with a reference-quality genome, single
nucleotide polymorphisms (SNPs) and short indels (insertions and deletions) can
be readily detected. Alignment of longer reads to a reference genome allows
detection of insertions, deletions, and inversions as well. When people say that the
human genome can now be sequenced for $1,000, they are referring to resequencing
of a human genome, not de novo sequencing and assembly of a human genome.
3.19 N50
N50 has become a common statistic used in describing genome assemblies. In my
opinion, most definitions of N50 are vague and rather difficult to comprehend.
However, through a diagram the idea of N50 can be relayed relatively easily. In
this regard, see Fig. 8.
3.20 Paired-End Sequencing and Unidirectional Sequencing
Paired-end sequencing (also known as bidirectional sequencing) is simply sequencing both ends of a DNA molecule. In some cases, paired-end sequencing provides as
much information and is considerably cheaper, than performing two unidirectional
(single-end) sequencing runs. In other instances, the paired-end data has value in
genome assembly.
The original paired-end sequencing was performed with cloned DNA molecules
and Sanger sequencing. The recombinant vector/insert molecules were isolated and
divided between two tubes. In the first tube, Sanger sequencing was performed using
a primer complementary to a piece of vector sequence immediately adjacent to one
side of the insertion site (forward sequencing reaction), while in the second tube, a
primer complementary to vector on the other side of the insertion site was used in
sequencing (reverse sequencing reaction).
9 Because new DNA strands are only
9 Forward and reverse designations are arbitrary. However, molecular biology companies often use
these designations to refer to specific vector-based primers, i.e., for paired-end sequencing or PCR
amplification using a particular vector, you will want to purchase both the forward and reverse
primers for that vector.
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D. G. Peterson and M. Arick
Resequencing is another technique that is poorly named as it typically does not refer
to sequencing the same DNA over again. Rather, resequencing is the quick sequencing of a genome via second- and/or third-generation techniques and alignment of the
resulting sequence reads to a reference-quality genome sequence (Lesk 2012).
Resequencing is used to quantify diversity and investigate changes in key genes.
When short-read sequences are aligned with a reference-quality genome, single
nucleotide polymorphisms (SNPs) and short indels (insertions and deletions) can
be readily detected. Alignment of longer reads to a reference genome allows
detection of insertions, deletions, and inversions as well. When people say that the
human genome can now be sequenced for $1,000, they are referring to resequencing
of a human genome, not de novo sequencing and assembly of a human genome.
3.19 N50
N50 has become a common statistic used in describing genome assemblies. In my
opinion, most definitions of N50 are vague and rather difficult to comprehend.
However, through a diagram the idea of N50 can be relayed relatively easily. In
this regard, see Fig. 8.
3.20 Paired-End Sequencing and Unidirectional Sequencing
Paired-end sequencing (also known as bidirectional sequencing) is simply sequencing both ends of a DNA molecule. In some cases, paired-end sequencing provides as
much information and is considerably cheaper, than performing two unidirectional
(single-end) sequencing runs. In other instances, the paired-end data has value in
genome assembly.
The original paired-end sequencing was performed with cloned DNA molecules
and Sanger sequencing. The recombinant vector/insert molecules were isolated and
divided between two tubes. In the first tube, Sanger sequencing was performed using
a primer complementary to a piece of vector sequence immediately adjacent to one
side of the insertion site (forward sequencing reaction), while in the second tube, a
primer complementary to vector on the other side of the insertion site was used in
sequencing (reverse sequencing reaction).
9 Because new DNA strands are only
9 Forward and reverse designations are arbitrary. However, molecular biology companies often use
these designations to refer to specific vector-based primers, i.e., for paired-end sequencing or PCR
amplification using a particular vector, you will want to purchase both the forward and reverse
primers for that vector.
140
D. G. Peterson and M. Arick