becomes an everyday research tool to address these questions [4]. Illumina NGS workflows
include the following basic steps:
• Library Preparation (see Chap. 3)
Libraries for NGS applications can be generated for diverse methods. Which library
preparation workflow to choose depends on your scientific or clinical question and its
relation to the genome, transcriptome, or epigenome of any organism. An overview of
the different Illumina Library Preparation Kits can be found at https://www.illumina.
com/products/by-type/sequencing-kits/library-prep-kits.html (see Chap. 3).
• Cluster Generation
Sequencing templates are immobilized on a flow cell surface designed to present the
DNA in a manner that facilitates access to enzymes while ensuring high stability of
surface-bound template and low non-specific binding of fluorescently labeled
nucleotides. Solid-phase amplification creates up to 1000 identical copies of each single
template molecule in close proximity.
• Sequencing
Illumina sequencing technology is also known as sequencing by synthesis (SBS)
technology. Four fluorescently labeled nucleotides are used to sequence the tens of
millions of clusters on the flow cell surface in parallel. During each sequencing cycle, a
single labeled deoxynucleoside triphosphate (dNTP) is added to the nucleic acid chain
and the nucleotide label serves as a reversible terminator for polymerization. After
removing the fluorescence label of previously attached dNTP another labeled dNTP is
added during a new sequencing cycle. Base calls are made directly from signal intensity
measurements during each cycle.
• Data Analysis
The NextSeq 550/2000, NextSeq 2000, and NovaSeq 6000 Sequencing Systems
generate raw data files in binary base call (BCL) format, requiring conversion to
FASTQ format for use with user-developed or third-party data analysis tools. Illumina
offers bcl2fastq Conversion Software to convert BCL files. bcl2fastq is an included,
standalone conversion software that demultiplexes data and converts BCL files to
standard FASTQ files, which are the starting format for data analysis.
As already described in Chap. 3, the library, which was prepared by random fragmentation of the DNA or cDNA (in terms of RNA-Seq) sample, followed by 5
0 and 3
0 adapter
ligation, PCR amplification, and gel purification (see Fig. 4.1a and Chap. 3). For cluster
generation, the library is loaded onto a flow cell where fragments are captured on a lawn of
surface-bound oligos complementary to the library adapters. Each fragment is then
amplified into distinct, clonal clusters through bridge amplification. When cluster generation is complete, the templates are ready for sequencing (Fig. 4.1b). The sequencing by
synthesis (SBS) technology uses a proprietary reversible terminator-based method that
4 NGS Technologies
49
include the following basic steps:
• Library Preparation (see Chap. 3)
Libraries for NGS applications can be generated for diverse methods. Which library
preparation workflow to choose depends on your scientific or clinical question and its
relation to the genome, transcriptome, or epigenome of any organism. An overview of
the different Illumina Library Preparation Kits can be found at https://www.illumina.
com/products/by-type/sequencing-kits/library-prep-kits.html (see Chap. 3).
• Cluster Generation
Sequencing templates are immobilized on a flow cell surface designed to present the
DNA in a manner that facilitates access to enzymes while ensuring high stability of
surface-bound template and low non-specific binding of fluorescently labeled
nucleotides. Solid-phase amplification creates up to 1000 identical copies of each single
template molecule in close proximity.
• Sequencing
Illumina sequencing technology is also known as sequencing by synthesis (SBS)
technology. Four fluorescently labeled nucleotides are used to sequence the tens of
millions of clusters on the flow cell surface in parallel. During each sequencing cycle, a
single labeled deoxynucleoside triphosphate (dNTP) is added to the nucleic acid chain
and the nucleotide label serves as a reversible terminator for polymerization. After
removing the fluorescence label of previously attached dNTP another labeled dNTP is
added during a new sequencing cycle. Base calls are made directly from signal intensity
measurements during each cycle.
• Data Analysis
The NextSeq 550/2000, NextSeq 2000, and NovaSeq 6000 Sequencing Systems
generate raw data files in binary base call (BCL) format, requiring conversion to
FASTQ format for use with user-developed or third-party data analysis tools. Illumina
offers bcl2fastq Conversion Software to convert BCL files. bcl2fastq is an included,
standalone conversion software that demultiplexes data and converts BCL files to
standard FASTQ files, which are the starting format for data analysis.
As already described in Chap. 3, the library, which was prepared by random fragmentation of the DNA or cDNA (in terms of RNA-Seq) sample, followed by 5
0 and 3
0 adapter
ligation, PCR amplification, and gel purification (see Fig. 4.1a and Chap. 3). For cluster
generation, the library is loaded onto a flow cell where fragments are captured on a lawn of
surface-bound oligos complementary to the library adapters. Each fragment is then
amplified into distinct, clonal clusters through bridge amplification. When cluster generation is complete, the templates are ready for sequencing (Fig. 4.1b). The sequencing by
synthesis (SBS) technology uses a proprietary reversible terminator-based method that
4 NGS Technologies
49