thus guarantees sequencing of the fragments. You can also ligate multiple indexing
adapters allowing to load more than one sample onto a flow-cell. This short index
enables you to distinguish between all the loaded samples carrying different indexing
adapters. Adapters have a defined length of ~60 bp, hence ~120 bp long fragments can
easily be identified as adapter dimers without any DNA/cDNA insert.
7. Purify Ligation Products (e.g., Pippin™ size selection; https://sagescience.com/
products/pippin-prep/).
This process purifies the products of the ligation reaction on a gel and removes
unligated adapters, as well as any adapters that might have ligated to one another.
8. Enrich DNA/cDNA Fragments and size selection via AMPure XP Beads.
This process uses PCR to selectively enrich those DNA/cDNA fragments that have
adapter molecules on both ends and to amplify the amount of DNA/cDNA in the library.
Additionally, it serves for size selection.
9. Validate Library and normalize and pool libraries.
This procedure is performed for quality control analysis on your sample library and
for quantification. Therefore, a Agilent Technologies Bioanalyzer or TapeStation
(https://www.agilent.com/en/product/automated-electrophoresis) and a Qubit 4 Fluorometer (https://www.thermofisher.com/de/de/home/industrial/spectroscopy-elemen
tal-isotope-analysis/molecular-spectroscopy/fluorometers/qubit/qubit-fluorometer.
html) are used.
This process describes how to prepare DNA/cDNA templates for cluster generation.
Indexed DNA/cDNA libraries are normalized to 10nM, and then pooled in equal
volumes.
Example library preparation protocols for RNA-Seq or ChIP-Seq, respectively, can be
found in the Appendix section (Sect. 13.1).
Take Home Message
• Different research or clinical questions require different library preparation
workflows.
• DNA sequencing approaches require proper fragmentation before library
preparation.
• RNA molecules are not directly sequenced due to their chemical instability and
the difficulty of processing and amplifying single-stranded nucleic acids. Thus,
RNA has to be converted into cDNA (reverse transcription) for sequencing
purpose.
• Quality and quantity determinations are essential within all library preparation
workflows.
3 Library Construction for NGS
43
adapters allowing to load more than one sample onto a flow-cell. This short index
enables you to distinguish between all the loaded samples carrying different indexing
adapters. Adapters have a defined length of ~60 bp, hence ~120 bp long fragments can
easily be identified as adapter dimers without any DNA/cDNA insert.
7. Purify Ligation Products (e.g., Pippin™ size selection; https://sagescience.com/
products/pippin-prep/).
This process purifies the products of the ligation reaction on a gel and removes
unligated adapters, as well as any adapters that might have ligated to one another.
8. Enrich DNA/cDNA Fragments and size selection via AMPure XP Beads.
This process uses PCR to selectively enrich those DNA/cDNA fragments that have
adapter molecules on both ends and to amplify the amount of DNA/cDNA in the library.
Additionally, it serves for size selection.
9. Validate Library and normalize and pool libraries.
This procedure is performed for quality control analysis on your sample library and
for quantification. Therefore, a Agilent Technologies Bioanalyzer or TapeStation
(https://www.agilent.com/en/product/automated-electrophoresis) and a Qubit 4 Fluorometer (https://www.thermofisher.com/de/de/home/industrial/spectroscopy-elemen
tal-isotope-analysis/molecular-spectroscopy/fluorometers/qubit/qubit-fluorometer.
html) are used.
This process describes how to prepare DNA/cDNA templates for cluster generation.
Indexed DNA/cDNA libraries are normalized to 10nM, and then pooled in equal
volumes.
Example library preparation protocols for RNA-Seq or ChIP-Seq, respectively, can be
found in the Appendix section (Sect. 13.1).
Take Home Message
• Different research or clinical questions require different library preparation
workflows.
• DNA sequencing approaches require proper fragmentation before library
preparation.
• RNA molecules are not directly sequenced due to their chemical instability and
the difficulty of processing and amplifying single-stranded nucleic acids. Thus,
RNA has to be converted into cDNA (reverse transcription) for sequencing
purpose.
• Quality and quantity determinations are essential within all library preparation
workflows.
3 Library Construction for NGS
43