Sequencing libraries
Boris Tichý
CF Genomics

Brno, 29.3.2021

Sequencing library
DNA fragments modified to allow unified access to fragments
Classic cDNA/DNA library
Cloning of DNA/cDNA into vectors (plasmid etc.)
NGS library
Adding of seuences that allow amplification (clonal) and sequencing

NGS libraries

Hundreds of types
Input
DNA, RNA, short RNA, crosslinked DNA/RNA
Adaptor adding strategy
Ligation, tagmentation, PCR
Specific sequence enrichment
Hybridization, PCR, imunoprecipitation

Library preparation

DNA fragmentation

Mechanical
Ultrasound (Covaris)
Hydrodynamic
Nebulization
Enzymatic
Restriction endonucleases
Fragmentase®
Transposase

Illumina
Cluster density depends on fragment length
Short fragments cluster better
Mix of longer and shorter fragments is problematic
Max <1000bp
Longer fragments are problematic
Broader length distribution =>
Uneven cluster generation effectivity
Problematic conc. measurment

Illumina NGS library

DNA

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Illumina NGS library - Ligation
3 steps:
End repair
A-tailing
Ligation (A-T)

Whole genomes
Enrichment libraries - input
Amplicons
ChIP, cDNA

Oligo B

Oligo B
A
T

DNA

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Random insert
Amplicon

T
A

Library preparation
Adaptor structure

Ligation

A) Full-length, usually single index

Complementary part (12bp)
T

Complementary part (12bp)

B) Short, usually dual index

Complementary part (12bp)
T

Complementary part (12bp)

Adaptor

PCR primers

Illumina NGS library - PCR

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DNA
Primer A

Primer B
Single genes/exons
Metagenomics – 16S
Smaller gene panels

Illumina NGS library – two-round PCR
Single genes/exons
Metagenomics – 16S
Smaller gene panels

Primer B - ext
Primer B - int

B

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Primer A - ext

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Primer A - int

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DNA

Illumina NGS library - tagmentation
Fragmentation and adaptor addition in single step
Transposase
Indexing with PCR

DNA

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Library preparation

Molecular barcodes
Tag each input molecule with random sequence before PCR amplification =>
Lower coverage for variant calling
Better quantification of variants (eg. species in metagenomics)
Haloplex HS

SAFE-Seq
First round – 2 cycles
ExoSAP treatment
Second round
Primer B - ext
Primer B – int with MB
DNA
Primer A – int with MB
Primer A - ext

Library preparation
Adaptor structure - UMI

Ligation

A) Full-length, usually single index

Complementary part (12bp)
T

Complementary part (12bp)
NNN

B) Short, usually dual index

NNN

Complementary part (12bp)

T NNN

Complementary part (12bp)

Adaptor

PCR primers