2.3 Sequencing platforms
The method that has been in place for the past few decades is Sanger
sequencing, this has been characterized by its simplicity but is capital
intensive and the running takes longer time than
necessary.33 Novel sequencing technologies that
immediately evolved after Sanger sequencing are collectively termed as
next-generation sequencing (NGS) and are often described as throughput,
accurate, cost-effective and reliable techniques that can examine the
whole genome within shortest period of time.33,34Generally, NGS platforms are grouped into second and third generations
but the most widely used platforms for second generation sequencing
include Ion Torrent and Illumina, on the contrary, Pacific Biosystems
and Oxford Nanopore Technologies are the most popular platforms for
third generation sequencing.35 Also, 454 FLX is among
the second generation platforms that was released in 2005 and works
based on pyrosequencing (i.e. sequencing-by-synthesis technique) like
Illumina.36 The most established Illumina platforms
include MiSeq, HiSeqs37 and NovaSeq, the newest
platform that is used for large-scale whole genome sequencing
analysis.38 MiSeq works as personalized sequencer that
sequence very small genomes with high speed and complete its operation
within 4 hours. On the other hand, HiSeqs such as HiSeq 2500 is designed
for “high-throughput” usage and mostly finish its cycle in 6 days
period.37 Unlike Illumina and 454 sequencing platforms
that are sequenced by synthesis, sequencing by oligonucleotide ligation
and detection (SOLiD) developed by Applied Biosystems (which later
became Life Technologies) is sequenced by ligation through hybridization
of short probes with the template DNA strand.36Despite its apparent advantages, its reads length and depth are not as
sophisticated as that of Illumina, thereby causing major assembly
challenge.39,40 Throughout all Illumina platforms,
only 1% error rate is recorded and substitution is regarded as their
major error,37 providing in depth sequencing capacity
that allows detection of very small quantity of transcripts in the
sample.41
Nevertheless, the third NGS sequencing platforms provide two essential
advantages over the second generation, including their ability to
increase reads length, avoid partiality of PCR in the amplification
process and the capacity to sequence single molecules at a given
time.36 Despite all advantages of NGS, the technique
has faced numerous challenges including numerous GC content, big size of
genome and the presence of homopolymers, however, various alternatives
have been put in place to overcome the current
challenges.28 Moreover, third NGS sequencing platforms
such as single-molecule real-time (SMRT) sequencing was developed by
Pacific Biosciences (PacBio), uses long reads that provides solution to
the myriads challenges faced by second generation platforms due to their
short reads length that make them unable to accurately detect gene
isoform, poor genome assembly and resolution of complicated genomic
region. On the contrary, PacBio sequencing (SMRT) is limited by high
percentage error, too costly per base and has
low-throughput.42 These challenges can be overcome by
the use of platforms with lower error rates as low as
3%38 and alternatively the use of hybrid Sanger
sequencing and PacBio sequencing technology have been
proposed.42 In addition, Helicos is one of the
single-molecule-based platforms with 5% error rate, reducing the number
of usable reads, making the reference genome extremely hard to be
matched with the sequence reads, and causing the loss of miRNA reads at
the alignment level.41 Due to their low error rate
(>1%), Illumina or SOLiD platforms are regarded as the
best alternative for miRNA sequencing because of its small
size.41 Nanopore sequencing platforms such as MinION,
PromethION and GridION37 belong to third generation
sequencing platforms developed by Oxford Nanopore Technologies, which
operates by passing a single stranded molecule of DNA or RNA via a
protein nanopore at the rate of 30 bases per seconds, through an
electrical current allowing direct sequencing of the molecule, thus
offering greater advantages.43 However, a major
setback of these platforms is having high error rate up to 10% to 20%
compare to other platforms with high-throughput.37,44
Most recently, several studies have reported remarkable achievements in
the diagnosis of different cancers including bladder cancer using
RNA-Seq.45-47 Transcriptomics (RNA-Seq) refers to the
application of any NGS platforms for the examination of
RNA41 and has become the best method for
whole-transcriptome profiling over the last decade.48The selection criteria for each platform depend on the purpose of the
experiments. The techniques operate similar to DNA sequencing except for
the library preparation and their data analysis which comprises assembly
of transcript, uncovering novel transcripts and calculation of
transcripts, among others.41 Additionally,
RNA-sequencing gives comprehensive, high-throughput, precise, accurate
and impartial view of the transcriptome analysis that overcome the
inherent limitations of real time PCR and microarray
techniques.49 These limitations include: the need for
previous knowledge of the sequence in question, inability to calculate
low and high expressed genes with great accuracy, among
others.22 RNA-Seq though highly throughput, it is too
expensive,50 especially for clinical settings.