456
information crucial for breeding programmes considering their long generation
times and long intervals of breeding cycle (Badenes et al. 2016). This chapter aims
to examine the state-of-the-art scientific knowledge on the application of HTS technologies on forest trees, particularly on genomic resources and marker development
for Argentinean native species.
17.1 High-Throughput Sequencing Technologies
The development of Sanger sequencing (Sanger et al. 1977) was the first step in recovering genomic information from different organisms. This is a method of DNA
sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by a DNA polymerase during in vitro DNA replication. The sequencing technology for the Sanger method has been progressively automated increasing the number
of samples that could be sequenced at once (Adams 2008; Heather and Chain 2016).
Therefore, Sanger technology has been widely adopted and employed in many largescale sequencing projects such as the human genome as well as model and non-model
species (e.g. Venter 2001; Chinwalla et al. 2002; Tuskan et al. 2006; Ueno et al. 2010).
After years of evolution, new high-throughput technologies emerged as the second generation of sequencing methods that exhibited better performance in aspects
that include massively parallel sequencing, high throughput and reduced cost (Liu
et al. 2012). The second-generation platforms vary with respect to throughput, cost,
read lengths which are generally short (average read length: 75–700 bp) and the
associated error rates varying according to platform and instrument (0,1-1) (Liu
et al. 2012; Kchouk et al. 2017; Pfeiffer et al. 2018). In general, massive parallelization is facilitated by the creation of many millions of individual reaction centres,
where DNA fragments are clonally amplified on a solid or liquid/emulsion medium
to be able then to distinguish signal from background noise. A sequencing platform
can collect information from all those reaction centres simultaneously and then
sequencing many millions of DNA molecules in parallel (Goodwin et al. 2016).
These HTS technologies are based on sequencing by ligation (SBL) and sequencing
by synthesis (SBS). Fundamentally, in SBL approaches, a probe sequence with a
fluorophore hybridizes to a DNA fragment and is ligated to an adjacent oligonucleotide for imaging. The emission spectrum of the different fluorophore probes gives
the sequences (SOLiD technology). In SBS a polymerase is used, and a signal identifies the incorporation of a nucleotide into an elongating strand. The signal could be
based on fluorescence (Illumina technology) which ranges from small to large ultrahigh- throughput ones (Reuter et al. 2015), change in ion (proton, H
+
) concentration
(Ion Torrent technology, Reuter et al. 2015) or bioluminescence (Roche/454 technology, Goodwin et al. 2016). The third-generation sequencing platforms, based on
generating longer DNA sequences, are single-molecule real-time (SMRT) developed by Pacific Biosciences (Roberts et al. 2013) and Oxford Nanopore Technologies
(Wang et al. 2015). These long-read sequencing technologies deliver reads in excess
of several kilobases (average 13–20 kb) allowing for the resolution of large structural features, such as complex or repetitive regions, and are also capable of
S. L. Torales et al.
information crucial for breeding programmes considering their long generation
times and long intervals of breeding cycle (Badenes et al. 2016). This chapter aims
to examine the state-of-the-art scientific knowledge on the application of HTS technologies on forest trees, particularly on genomic resources and marker development
for Argentinean native species.
17.1 High-Throughput Sequencing Technologies
The development of Sanger sequencing (Sanger et al. 1977) was the first step in recovering genomic information from different organisms. This is a method of DNA
sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by a DNA polymerase during in vitro DNA replication. The sequencing technology for the Sanger method has been progressively automated increasing the number
of samples that could be sequenced at once (Adams 2008; Heather and Chain 2016).
Therefore, Sanger technology has been widely adopted and employed in many largescale sequencing projects such as the human genome as well as model and non-model
species (e.g. Venter 2001; Chinwalla et al. 2002; Tuskan et al. 2006; Ueno et al. 2010).
After years of evolution, new high-throughput technologies emerged as the second generation of sequencing methods that exhibited better performance in aspects
that include massively parallel sequencing, high throughput and reduced cost (Liu
et al. 2012). The second-generation platforms vary with respect to throughput, cost,
read lengths which are generally short (average read length: 75–700 bp) and the
associated error rates varying according to platform and instrument (0,1-1) (Liu
et al. 2012; Kchouk et al. 2017; Pfeiffer et al. 2018). In general, massive parallelization is facilitated by the creation of many millions of individual reaction centres,
where DNA fragments are clonally amplified on a solid or liquid/emulsion medium
to be able then to distinguish signal from background noise. A sequencing platform
can collect information from all those reaction centres simultaneously and then
sequencing many millions of DNA molecules in parallel (Goodwin et al. 2016).
These HTS technologies are based on sequencing by ligation (SBL) and sequencing
by synthesis (SBS). Fundamentally, in SBL approaches, a probe sequence with a
fluorophore hybridizes to a DNA fragment and is ligated to an adjacent oligonucleotide for imaging. The emission spectrum of the different fluorophore probes gives
the sequences (SOLiD technology). In SBS a polymerase is used, and a signal identifies the incorporation of a nucleotide into an elongating strand. The signal could be
based on fluorescence (Illumina technology) which ranges from small to large ultrahigh- throughput ones (Reuter et al. 2015), change in ion (proton, H
+
) concentration
(Ion Torrent technology, Reuter et al. 2015) or bioluminescence (Roche/454 technology, Goodwin et al. 2016). The third-generation sequencing platforms, based on
generating longer DNA sequences, are single-molecule real-time (SMRT) developed by Pacific Biosciences (Roberts et al. 2013) and Oxford Nanopore Technologies
(Wang et al. 2015). These long-read sequencing technologies deliver reads in excess
of several kilobases (average 13–20 kb) allowing for the resolution of large structural features, such as complex or repetitive regions, and are also capable of
S. L. Torales et al.