12 research outputs found
Comparing Three Approaches
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
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 â
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 â 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 KontiguitÀt-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 wÀhrend 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
EinschrÀnkungen 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 wÀhrend des
gesamten Prozesses zur Vorbereitung der Sequenzierungsbibliotheken zu
bewahren. Das entwickelte Protokoll enthÀlt detaillierte Anweisungen zum
Steuerungs-Setup und Richtlinien fĂŒr die anschlieĂende 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 hÀngt 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
<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.
<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 ESâ=âSt; ThruPLEX-FD ESâ=âTp; REPLI-g ESâ=âRg; GenomePlex ESâ=âGp. The lower left tile presents the overall statistics, where âTotalâ 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
Coverage distribution along the target regions with different percentages of GC bases.
<p>Coverage distribution along the target regions with different percentages of GC bases.</p
Alignment statistics.
<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
The experimental scheme.
<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.
<p>Analysis was performed on subsets of reads uniquely mapped to the target region and having approximately equal total amounts of bases (<b>âŒ</b>17<b>Ă</b>10<sup>8</sup> bases).</p
Per-base sequencing depth distribution on the target region.
<p>Per-base sequencing depth distribution on the target region.</p