12 research outputs found

    Comparing Three Approaches

    Get PDF
    Hybridization-based target enrichment protocols require relatively large starting amounts of genomic DNA, which is not always available. Here, we tested three approaches to pre-capture library preparation starting from 10 ng of genomic DNA: (i and ii) whole-genome amplification of DNA samples with REPLI-g (Qiagen) and GenomePlex (Sigma) kits followed by standard library preparation, and (iii) library construction with a low input oriented ThruPLEX kit (Rubicon Genomics). Exome capture with Agilent SureSelectXT2 Human AllExon v4+UTRs capture probes, and HiSeq2000 sequencing were performed for test libraries along with the control library prepared from 1 ”g of starting DNA. Tested protocols were characterized in terms of mapping efficiency, enrichment ratio, coverage of the target region, and reliability of SNP genotyping. REPLI-g- and ThruPLEX-FD-based protocols seem to be adequate solutions for exome sequencing of low input sample

    Von Tn5-basierter DNS-Fragmentierungseffizienz und Beurteilung des 'tagmentation site indexing'-Ansatzes fĂŒr die kontiguitĂ€t-bewahrende Sequenzierung

    No full text
    ACKNOWLEDGMENTS 6 ABBREVIATIONS 7 SUMMARY 8 Zusammenfassung 9 INTRODUCTION 10 Sequencing gaps: loss of contiguity information 10 Solutions for contiguity preserving sequencing 13 Extending the read length: single molecule sequencing 13 Experimental approaches to link short reads 16 Subselection of a particular part of a genome 16 Random subselection of a part of a genome 20 Cloning pools 20 Fragment pools 22 CPT-seq 24 Proximity ligation strategies 27 Paired indexing of fragmentation sites 29 MATERIALS AND METHODS 34 Materials 34 Chemicals 34 Hardware and Plastic 36 Enzymes 38 Kits 39 Oligonucleotides 39 Plasmids 43 Solutions 43 Methods 45 Tn5 Production 45 Bacterial Stab 45 Glycerol Stocks 45 Plasmid DNA Preparation 45 Sequencing 46 Protein expression 46 Transformation T7 Express lysY/Iq Competent E. coli cells 46 Overexpression 46 Tn5 purification 47 Preparation of Chitin Magnetic Beads 47 Binding 47 Cleavage 48 Elution 48 Protein concentration and buffer exchange 49 Transposase activity assay 49 Fragmentation Efficiency Assay (FEA) 50 Transposon 50 Transposome assembly 50 Tagmentation template 51 Tagmentation 51 qPCR 52 Visual fragments size analysis 52 PITS Library Preparation 53 Transposome assembly 53 Phage Lambda genomic DNA 53 Tagmentation 53 Dilution 54 Oligo replacement and gap-filling reaction 54 Amplification 55 Flow cell loading and sequencing 56 Sequencing analysis 56 Preparation of Alternative Transposon structures 57 Synthetic Lampion Transposon 57 Preparation of PCR Products of Different Lengths 57 Transposon Insertion with Epicentre Transposase 58 Transposon Insertion with In-House Tn5 Transposase 59 RESULTS AND DISCUSSION 60 Preface 60 Tagmentation sites indexing approach for contiguity-preserving sequencing 60 NGS library preparation from amol amounts of DNA and non-residual loading on Illumina sequencing flowcell 61 Tagmentation 63 Dilution of the tagmentation reaction 64 Gap repair reaction 65 Amplification and loading on a flowcell 67 Recommended experimental setup 68 Proof-of principle paired indexing experiment 71 Indexing scheme 71 Template selection 73 PITS libraries sequencing 73 Preparation of in-house Tn5 Transposase 78 Tn5 Transposase expression and purification 78 Transposase activity assay 79 Fragmentation efficiency assay (FEA) 79 Discussion of the PITS protocol 86 REFERENCES 89 SUPPLEMENTARY 100A novel contiguity-preserving sequencing approach was developed and tested – paired indexing at tagmentation sites (PITS). The idea of the method is to use paired indices to individually label pairs of DNA molecule ends originating at tagmentation sites. Indices from the same pair on the ends of two sequencing library fragments would mean that those fragments were next to each other in the original molecule. Thus after getting separated in the way it occurs in a standard sequencing library preparation protocol library, fragments after sequencing would be assembled again and the contiguity of the sequence would be restored. Convenient system for testing of the PITS method was chosen and proof-of-principle experiment was performed. Though few, scaffolds containing up to 4 subsequent library molecules were assembled. Some of the constraints of the PITS approach were revealed and optimization possibilities for future work were determined. A patent application describing the PITS protocol is in preparation. During the course of this PhD, an efficient method for preparation of sequencing library from amol amounts of tagmentation products has been established – post tagmentation ultra low input (PTULI) protocol. The method aims at preserving possibly all tagmentation fragments throughout sequencing library preparation process. The developed protocol provides detailed instructions on the controls setup and guidelines for subsequent non- residual loading of the PTULI library on a sequencer. The PTULI library preparation strategy is suitable for PITS, and is also of value for other minute input material sequencing applications. To make contiguity-preserving sequencing work possible, in-house Tn5 transposase was prepared. Applications of this enzyme within the settings other than those in commercially available kits are hardly known and further development of the PITS approach to a great extent depends on the potential of this enzyme. That is why in parallel to the PITS method itself, Tn5 properties were studied. An electrophoresis free assay for characterizing Tn5 transposomes fragmentation efficiency was developed and published [Rykalina et al., 2017]. This assay is a convenient monitoring system for the setup of tagmentation protocols and for optimization experiments.Es wurde ein neuartiger KontiguitĂ€t-bewahrender Sequenzierungsansatz entwickelt und getestet: gepaarte Indizierung an Tagmentationsstellen (Paired Indexing at Tagmentation Sites - PITS). Die Idee der Methode besteht darin, die Paare der DNA-MolekĂŒl-Enden, die ursprĂŒnglich von Tagmentationsstellen stammen, individuell mit gepaarten Indizes zu markieren. Gleiche Indizes an den Enden der zwei sequenzierten Bibliotheksfragmente wĂŒrde bedeuten, dass diese Fragmente im ursprĂŒnglichen MolekĂŒl nebeneinander angeordnet waren. Dank dieser Methode werden die Fragmente, die wĂ€hrend der Herstellung von Sequenzierungsbibliotheken getrennt wurden, wieder zusammengesetzt und damit die KontiguitĂ€t der Sequenz der ursprĂŒnglichen DNA-MolekĂŒle wiederhergestellt. Es wurde ein praktisches System fĂŒr die PrĂŒfung der PITS-Methode gewĂ€hlt und ein proof-of-principle Experiment durchgefĂŒhrt. DNA Ketten mit bis zu vier nachfolgenden BibliotheksmolekĂŒlen wurden zusammengebaut. Einige der EinschrĂ€nkungen des PITS-Ansatzes wurden aufgedeckt und Optimierungsmöglichkeiten fĂŒr zukĂŒnftige Arbeiten ermittelt. Eine Patentanmeldung, die das PITS-Protokoll beschreibt, ist in Vorbereitung. Im Rahmen der Arbeit wurde ein effizientes Verfahren zur Herstellung von Sequenzierungsbibliotheken aus amol-Mengen von Tagmentationsprodukten etabliert, das Post-Tagmentation Ultra Low Input (PTULI) Protokoll. Die Methode zielt darauf ab, möglichst alle Tagmentierungsfragmente wĂ€hrend des gesamten Prozesses zur Vorbereitung der Sequenzierungsbibliotheken zu bewahren. Das entwickelte Protokoll enthĂ€lt detaillierte Anweisungen zum Steuerungs-Setup und Richtlinien fĂŒr die anschließende komplette Beladung der PTULI-Bibliothek auf einem Sequenzer. Die PTULI- Bibliotheksvorbereitungsstrategie eignet sich nicht nur fĂŒr PITS, sondern auch fĂŒr andere Sequenzierungsanwendungen mit geringen Input. Um dieses Projekt zu ermöglichen, wurde eine eigene Tn5-Transposase hergestellt. Anwendungen dieses Enzyms in anderen settings als in handelsĂŒblichen Kits sind kaum bekannt und die Weiterentwicklung des PITS-Ansatzes hĂ€ngt weitgehend von dem Potenzial dieses Enzyms ab. Deshalb wurden parallel zur PITS-Methode selbst Tn5-Eigenschaften untersucht. Ein elektrophoresefreier Assay zur Charakterisierung von Tn5-Transposomen Fragmentierungseffizienz wurde entwickelt und veröffentlicht [Rykalina et al., 2017]. Dieser Assay bietet ein bequemes Monitoring-System fĂŒr den Aufbau von Tagmentationsprotokollen und fĂŒr Optimierungsexperimente

    Exome Sequencing from Nanogram Amounts of Starting DNA: Comparing Three Approaches

    No full text
    <div><p>Hybridization-based target enrichment protocols require relatively large starting amounts of genomic DNA, which is not always available. Here, we tested three approaches to pre-capture library preparation starting from 10 ng of genomic DNA: (i and ii) whole-genome amplification of DNA samples with REPLI-g (Qiagen) and GenomePlex (Sigma) kits followed by standard library preparation, and (iii) library construction with a low input oriented ThruPLEX kit (Rubicon Genomics). Exome capture with Agilent SureSelect<i><sup>XT2</sup></i> Human AllExon v4+UTRs capture probes, and HiSeq2000 sequencing were performed for test libraries along with the control library prepared from 1 ”g of starting DNA. Tested protocols were characterized in terms of mapping efficiency, enrichment ratio, coverage of the target region, and reliability of SNP genotyping. REPLI-g- and ThruPLEX-FD-based protocols seem to be adequate solutions for exome sequencing of low input samples.</p></div

    Sharing of genetic variations between strategies depicted in a Venn diagram.

    No full text
    <p>Only variation with minimum depth of coverage of 20x and minimum quality of 13 were taken into account in all four strategies. The names of the samples are abbreviated: Standard ES = St; ThruPLEX-FD ES = Tp; REPLI-g ES = Rg; GenomePlex ES = Gp. The lower left tile presents the overall statistics, where “Total” indicates the number of all unique SNVs found in the region of interest, i.e. the union of SNV sets found by each strategy.</p

    The experimental scheme.

    No full text
    <p>Two DNA samples (Test DNA 1 and Test DNA 2) were subjected to four exome sequencing (ES) protocols performed in parallel: control (Standard ES) and three modified (REPLI-g ES, GenomePlex ES and ThruPLEX-FD ES). Common steps performed in parallel for several protocols are shown by text boxes spanning the corresponding number of protocol columns.</p

    Coverage statistics for the target region.

    No full text
    <p>Analysis was performed on subsets of reads uniquely mapped to the target region and having approximately equal total amounts of bases (<b>∌</b>17<b>×</b>10<sup>8</sup> bases).</p

    Alignment statistics.

    No full text
    <p>*high confident reads-reads with probability of wrong mapping lower than 0.05 according to their MAPQ score (MAPQ>13).</p><p>**some of GenomePlex ES library reads contained sequences of the primer used for whole genome amplification. These common segments were cut out before the alignment. As a result, 13.8% of and 11.9% of nucleotides were removed from the reads of the Test DNA 1 and Test DNA 2 libraries, respectively.</p><p>***FR-flanking regions (FR), which include 100<b> </b>bp from both ends of the targeted sequences.</p
    corecore