3.1
Introduction
Library preparation involves generating a collection of DNA/cDNA fragments for
sequencing. NGS libraries are typically prepared by fragmenting a DNA or RNA sample
and ligating specialized adapters to both fragments ends. In this textbook we will focus on
the Illumina
® Library Preparation workflow for short-read sequencing (https://emea.
illumina.com/techniques/sequencing/ngs-library-prep.html). As already mentioned, a wide
variety of NGS methods exists, and for almost every method a comprehensive sequencing
library preparation solution. The principle is almost similar for all library preparation
workflows [1, 2]. Further information on library preparation for long-read [3] or singlecell sequencing [4–6] can be found on the websites of the corresponding providers:
Single-cell sequencing
• Illumina (https://emea.illumina.com/techniques/sequencing/rna-sequencing/ultra-lowinput-single-cell-rna-seq.html)
• 10xGenomics (https://support.10xgenomics.com/single-cell-gene-expression/
automated-library-prep)
• Qiagen (https://www.qiagen.com/de/products/discovery-and-translational-research/
next-generation-sequencing/library-preparation/qiaseq-fx-single-cell-dna-library-kit/
#orderinginformation)
• And many others
Long-read sequencing:
• Illumina (https://emea.illumina.com/science/technology/next-generation-sequencing/
long-read-sequencing.html)
• PacificBioscience (https://www.pacb.com/products-and-services/consumables/tem
plate-preparation-multiplexing-kits/)
• Oxford Nanopore (https://nanoporetech.com/products/kits)
• And many others
3.2
Library Preparation Workflow
The core steps in preparing RNA or DNA for NGS are:
• Fragmenting and/or sizing the target sequences to a desired length.
• Converting target to double-stranded DNA (in terms of RNA-Seq).
• Attaching oligonucleotide adapters to the ends of target fragments.
• Quantifying the final library product for sequencing.
40
M. Kappelmann-Fenzl
Introduction
Library preparation involves generating a collection of DNA/cDNA fragments for
sequencing. NGS libraries are typically prepared by fragmenting a DNA or RNA sample
and ligating specialized adapters to both fragments ends. In this textbook we will focus on
the Illumina
® Library Preparation workflow for short-read sequencing (https://emea.
illumina.com/techniques/sequencing/ngs-library-prep.html). As already mentioned, a wide
variety of NGS methods exists, and for almost every method a comprehensive sequencing
library preparation solution. The principle is almost similar for all library preparation
workflows [1, 2]. Further information on library preparation for long-read [3] or singlecell sequencing [4–6] can be found on the websites of the corresponding providers:
Single-cell sequencing
• Illumina (https://emea.illumina.com/techniques/sequencing/rna-sequencing/ultra-lowinput-single-cell-rna-seq.html)
• 10xGenomics (https://support.10xgenomics.com/single-cell-gene-expression/
automated-library-prep)
• Qiagen (https://www.qiagen.com/de/products/discovery-and-translational-research/
next-generation-sequencing/library-preparation/qiaseq-fx-single-cell-dna-library-kit/
#orderinginformation)
• And many others
Long-read sequencing:
• Illumina (https://emea.illumina.com/science/technology/next-generation-sequencing/
long-read-sequencing.html)
• PacificBioscience (https://www.pacb.com/products-and-services/consumables/tem
plate-preparation-multiplexing-kits/)
• Oxford Nanopore (https://nanoporetech.com/products/kits)
• And many others
3.2
Library Preparation Workflow
The core steps in preparing RNA or DNA for NGS are:
• Fragmenting and/or sizing the target sequences to a desired length.
• Converting target to double-stranded DNA (in terms of RNA-Seq).
• Attaching oligonucleotide adapters to the ends of target fragments.
• Quantifying the final library product for sequencing.
40
M. Kappelmann-Fenzl