than 2 kb can cause problems with bridge amplification,
10 so paired-end Illumina
sequencing is currently limited to target molecules <2 kb in length.
For Illumina platforms, paired-end sequencing is often indicated by the number
two followed by a multiplication sign followed by read length in bp or nt. Thus, if
you order 2 Â 250 nt sequencing from a vendor, you are requesting paired-end
sequencing with 250 nt reads (500 nt total sequence per molecule). If sequencing is
unidirectional, it is denoted by the read length alone or by the number one times the
read length (i.e., 250 bp or 1 Â 250 bp).
3.21 Mate-Pair Sequencing
With the exception of Illumina, simple paired-end sequencing is not an option for
second-generation techniques. However, there is a technique known as mate-pair
sequencing that can be used to sequence the ends of fairly large DNA molecules.
Mate-pair approaches involve adding biotin or biotinylated adapters to the ends of
fairly long sequences, circularization of DNA molecules, fragmentation of circularized molecules to a size suitable for sequencing on the platform of choice, and
enrichment of biotin-containing fragments using streptavidin. Typically, regular
sequencing adapters are added to the ends of the mate-pair molecules. Procedures
for making mate-pair libraries can be quite sophisticated. For example, the mate-pair
library technique of Jarvie and Harkins (2008), developed for 454 sequencing,
involves shearing DNA into 3 kb fragments, methylating the DNA fragments
(to prevent restriction later on), ligating a biotinylated hairpin adapter to both ends
of each fragment, digestion of the hairpin adapters with a restriction enzyme that
does not cut methylated DNA (e.g., EcoRI) to produce complementary sticky ends,
ligation of ends together to create circularized molecules, shearing of the circularized
DNA into 250 bp fragments, streptavidin-based isolation of fragments containing the
remainder of the two annealed hairpin adapters, and addition of A and B adapters for
emulsion PCR amplification and sequencing.
PacBio and Oxford Nanopore produce long-read lengths from single DNA
molecules not fixed to a substrate. Consequently, producing paired-end reads or
mate-pair sequences is not a goal of either of these methods.
3.22 Multiplexing, Indexing, and Bar Coding
Second-generation sequencers can produce so many reads that it is often economical
to group different DNA libraries together before sequencing them on a single lane/
10 This is according to Illumina; our own experience suggests a dramatic decrease in quality if target
molecule length exceeds 900 bp.
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D. G. Peterson and M. Arick
10 so paired-end Illumina
sequencing is currently limited to target molecules <2 kb in length.
For Illumina platforms, paired-end sequencing is often indicated by the number
two followed by a multiplication sign followed by read length in bp or nt. Thus, if
you order 2 Â 250 nt sequencing from a vendor, you are requesting paired-end
sequencing with 250 nt reads (500 nt total sequence per molecule). If sequencing is
unidirectional, it is denoted by the read length alone or by the number one times the
read length (i.e., 250 bp or 1 Â 250 bp).
3.21 Mate-Pair Sequencing
With the exception of Illumina, simple paired-end sequencing is not an option for
second-generation techniques. However, there is a technique known as mate-pair
sequencing that can be used to sequence the ends of fairly large DNA molecules.
Mate-pair approaches involve adding biotin or biotinylated adapters to the ends of
fairly long sequences, circularization of DNA molecules, fragmentation of circularized molecules to a size suitable for sequencing on the platform of choice, and
enrichment of biotin-containing fragments using streptavidin. Typically, regular
sequencing adapters are added to the ends of the mate-pair molecules. Procedures
for making mate-pair libraries can be quite sophisticated. For example, the mate-pair
library technique of Jarvie and Harkins (2008), developed for 454 sequencing,
involves shearing DNA into 3 kb fragments, methylating the DNA fragments
(to prevent restriction later on), ligating a biotinylated hairpin adapter to both ends
of each fragment, digestion of the hairpin adapters with a restriction enzyme that
does not cut methylated DNA (e.g., EcoRI) to produce complementary sticky ends,
ligation of ends together to create circularized molecules, shearing of the circularized
DNA into 250 bp fragments, streptavidin-based isolation of fragments containing the
remainder of the two annealed hairpin adapters, and addition of A and B adapters for
emulsion PCR amplification and sequencing.
PacBio and Oxford Nanopore produce long-read lengths from single DNA
molecules not fixed to a substrate. Consequently, producing paired-end reads or
mate-pair sequences is not a goal of either of these methods.
3.22 Multiplexing, Indexing, and Bar Coding
Second-generation sequencers can produce so many reads that it is often economical
to group different DNA libraries together before sequencing them on a single lane/
10 This is according to Illumina; our own experience suggests a dramatic decrease in quality if target
molecule length exceeds 900 bp.
142
D. G. Peterson and M. Arick