The preparation of a high-quality sequencing library plays an important role in NextGeneration Sequencing (NGS). The first major step in preparing nucleic acids for NGS is
fragmentation. The most common and effective fragmentation methods can be subdivided
into three classes:
1. Physical fragmentation (are acoustic shearing, sonication, and hydrodynamic shear).
2. Enzymatic fragmentation (DNase I or other restriction endonuclease, non-specific
nuclease, Transposase).
3. Chemical fragmentation (heat and divalent metal cation). This method is used to break
up long RNA fragments, whereas the length of your RNA can be adjusted by
modulating the incubation time.
But also, a PCR amplification of genetic loci of interest can be chosen. Each NGS
approach has its own specific protocol. Available NGS sample preparation kits are:
Illumina, New England BioLabs, KAPA Biosystems, Swift Bioscience, Enzymatics,
BIOO, etc. The principle workflow after fragmentation can be briefly described by the
following working steps, which are also illustrated in a simplified way in Fig. 3.1 (the
numbering of each working step is analog to the numbering in Fig. 3.1):
1. Quantification and profile your isolated DNA or RNA samples. This is one of the most
important steps after sample preparation. For sequencing the samples have to be of a
very good quality, the concentration must be determined, and the fragmentation efficiency must be checked (target size for short-read sequencing is commonly 200bp–
800bp) before you can go any further.
2. Perform End Repair and size selection via AMPure XP Beads (Beckman Coulter
Genomics [7]). This process converts the overhangs resulting from fragmentation into
blunt ends and serves for size selection. End Repair is not performed during RNA-Seq
library preparation.
3. In Terms of RNA-Seq Library Preparation [8].
Depletion of rRNA and fragmentation or polyA-capture or another procedure for
isolating the RNA type of interest (depending on your research question and library
preparation kit you use: https://emea.illumina.com/products/by-type/sequencing-kits/
library-prep-kits.html) is necessary.
4. The RNA fragments obtained must then be transcribed into cDNA for sequencing by
synthesis of the first cDNA strand followed by synthesis of the second cDNA strand.
5. Adenylate 3’-Ends of DNA/cDNA.
A single “A” nucleotide is added to the 3’ ends of the blunt fragments to prevent them
from ligating to one another during the adapter ligation reaction. A corresponding single
“T” nucleotide on the 3’ end of the adapter provides a complementary overhang for
ligating the adapter to the fragment.
6. Adapter Ligation and Size Selection via AMPure XP Beads of DNA/cDNA
Adapter ligation is a crucial step within the NGS library preparation. Adapters are
design with a single “T” nucleotide on the 3’ end to recognize the “A” nucleotide
3 Library Construction for NGS
41
fragmentation. The most common and effective fragmentation methods can be subdivided
into three classes:
1. Physical fragmentation (are acoustic shearing, sonication, and hydrodynamic shear).
2. Enzymatic fragmentation (DNase I or other restriction endonuclease, non-specific
nuclease, Transposase).
3. Chemical fragmentation (heat and divalent metal cation). This method is used to break
up long RNA fragments, whereas the length of your RNA can be adjusted by
modulating the incubation time.
But also, a PCR amplification of genetic loci of interest can be chosen. Each NGS
approach has its own specific protocol. Available NGS sample preparation kits are:
Illumina, New England BioLabs, KAPA Biosystems, Swift Bioscience, Enzymatics,
BIOO, etc. The principle workflow after fragmentation can be briefly described by the
following working steps, which are also illustrated in a simplified way in Fig. 3.1 (the
numbering of each working step is analog to the numbering in Fig. 3.1):
1. Quantification and profile your isolated DNA or RNA samples. This is one of the most
important steps after sample preparation. For sequencing the samples have to be of a
very good quality, the concentration must be determined, and the fragmentation efficiency must be checked (target size for short-read sequencing is commonly 200bp–
800bp) before you can go any further.
2. Perform End Repair and size selection via AMPure XP Beads (Beckman Coulter
Genomics [7]). This process converts the overhangs resulting from fragmentation into
blunt ends and serves for size selection. End Repair is not performed during RNA-Seq
library preparation.
3. In Terms of RNA-Seq Library Preparation [8].
Depletion of rRNA and fragmentation or polyA-capture or another procedure for
isolating the RNA type of interest (depending on your research question and library
preparation kit you use: https://emea.illumina.com/products/by-type/sequencing-kits/
library-prep-kits.html) is necessary.
4. The RNA fragments obtained must then be transcribed into cDNA for sequencing by
synthesis of the first cDNA strand followed by synthesis of the second cDNA strand.
5. Adenylate 3’-Ends of DNA/cDNA.
A single “A” nucleotide is added to the 3’ ends of the blunt fragments to prevent them
from ligating to one another during the adapter ligation reaction. A corresponding single
“T” nucleotide on the 3’ end of the adapter provides a complementary overhang for
ligating the adapter to the fragment.
6. Adapter Ligation and Size Selection via AMPure XP Beads of DNA/cDNA
Adapter ligation is a crucial step within the NGS library preparation. Adapters are
design with a single “T” nucleotide on the 3’ end to recognize the “A” nucleotide
3 Library Construction for NGS
41