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 DNA fragmentation Library preparation 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 DNA AmplificationadaptorA Barcode/indexA SequencingprimerA SequencingprimerB AmplificationadaptorB Barcode/indexB Illumina NGS library DNA AmplificationadaptorA Barcode/indexA SequencingprimerA SequencingprimerB AmplificationadaptorB Barcode/indexB Illumina NGS library - Ligation Oligo BOligo B Random insert Amplicon A T T A 3 steps: End repair A-tailing Ligation (A-T) Whole genomes Enrichment libraries - input Amplicons ChIP, cDNA Library preparation LigationAdaptor structure Complementary part (12bp) Complementary part (12bp)T A) Full-length, usually single index B) Short, usually dual index Complementary part (12bp) Complementary part (12bp)T Adaptor PCR primers DNA AmplificationadaptorA Barcode/indexA SequencingprimerA SequencingprimerB AmplificationadaptorB Barcode/indexB Illumina NGS library - PCR Primer A Primer B Single genes/exons Metagenomics – 16S Smaller gene panels DNA AmplificationadaptorA Barcode/indexA SequencingprimerA SequencingprimerB AmplificationadaptorB Barcode/indexB Illumina NGS library – two-round PCR Primer A - int Primer B - ext Primer B - int Primer A - ext Single genes/exons Metagenomics – 16S Smaller gene panels DNA AmplificationadaptorA Barcode/indexA SequencingprimerA SequencingprimerB AmplificationadaptorB Barcode/indexB Illumina NGS library - tagmentation Fragmentation and adaptor addition in single step Transposase Indexing with PCR Molecular barcodes Library preparation 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 DNA Primer A – int with MB Primer B - ext Primer B – int with MB Primer A - ext First round – 2 cycles ExoSAP treatment Second round Library preparation LigationAdaptor structure - UMI Complementary part (12bp) Complementary part (12bp)T A) Full-length, usually single index B) Short, usually dual index Complementary part (12bp) Complementary part (12bp)T Adaptor PCR primers NNN NNN NNN