Various sources of extracted genomic DNA (gDNA) were used for amplification (Supplementary Figure S1 ). (i) Whole worm: one worm or 10 adult worms were cut into pieces with a 22-gauge needle in a 200 μl PCR tube. In one amplification instance, a single whole worm was prepared with 5% dimethylsulfoxide (DMSO) added in polymerase mix. Genomic DNA extraction and amplification were performed with the Qiagen REPLI-g Kit (150023,150043,150343, Qiagen, German). (ii) Purified DNA: single-worm DNA extraction with lysis buffer [8 μl of direct PCR lysis reagent (Viagen, #102-T), 1 μl of 5 mg/ml proteinase K, 1 μl of 200 mM DTT) and incubated in 65°C for 20 min and 95°C for 5 min. The samples were further purified using a 1:1 v/v ratio of sample to Ampure XP beads (A63882, Beckman Coulter, USA) and eluted in 10 μl of elution buffer. The MDA step was performed with the REPLI-g Kit (150023, Qiagen) following the manufacturer's instructions. A detailed protocol is available at protocols.io .
Repli g kit
Manufactured by Qiagen
Sourced in Germany, United States
The REPLI-g kit is a genomic DNA amplification product from Qiagen. It is designed for whole genome amplification (WGA) from a small number of cells or limited amounts of DNA.
Automatically generated - may contain errors
Lab products found in correlation
Dneasy blood and tissue kit, by Qiagen (4 mentions)
Qiaamp dna mini kit, by Qiagen (3 mentions)
Nucleospin blood quickpure kit, by Macherey-Nagel (2 mentions)
Seqcap ez human exome library v3, by Agilent Technologies (2 mentions)
Agilent human exon v3 exon 50mb, by Agilent Technologies (2 mentions)
Sureselect human all exon v4, by Agilent Technologies (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (2 mentions)
Catch all collection swabs, by Illumina (2 mentions)
Buccalamp dna extraction kit, by Illumina (2 mentions)
Wizard dna extraction kit, by Promega (2 mentions)
Rneasy extraction kit, by Qiagen (2 mentions)
Primestar gxl dna polymerase, by Takara Bio (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Repli g single cell kit, by Qiagen (2 mentions)
Agencourt ampure xp bead, by Beckman Coulter (2 mentions)
Repli g mini kit, by Qiagen (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Dna ffpe tissue kit, by Qiagen (2 mentions)
Nucleospin tissue xs, by Macherey-Nagel (1 mentions)
61 protocols using repli g kit
1
Whole Worm DNA Extraction and Amplification
2
DNA Extraction and Amplification Protocol
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DNA was isolated using NucleoSpin Blood QuickPure kit (Macherey Nagel, Düren, Germany) or NucleoSpin Tissue XS (Macherey Nagel, Düren, Germany), according to the manufacturer’s protocol. When the extraction yield was insufficient (< 5 µg), 5 µl of DNA was amplified using the Qiagen REPLI-g kit (Qiagen, Venlo, The Netherlands) according to the manufacturer’s protocol. All amplifications were performed in duplicate. The detailed protocol can be found in the supplementary materials and methods .
3
Isolation and Amplification of DNA from Myelodysplastic Syndrome Samples
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DNA was isolated from PB or BM of MDS patients using the NucleoSpin Blood QuickPure kit (Macherey Nagel, Düren, Germany) according to the manufacturer’s protocol. In addition, BM and PB mononuclear cells (MNCs) and PB granulocytes were obtained after Ficoll-1077 density gradient separation. BM or PB cells were slowly added on top of a layer with Ficoll-Paque PLUS (density 1.077) (GE Healthcare, Chicago, IL, USA). After centrifugation at 700 g for 20 min, MNCs were present on top of the Ficoll layer and granulocytes (and red bloods) underneath. These two cell fractions were collected separately, after which DNA was isolated. When the extraction yield was insufficient (<5 μg) as measured with the Qubit fluorometer Quant-iT dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), 80 ng of DNA was amplified using the Qiagen REPLI-g kit (Qiagen, Venlo, The Netherlands) in 4 parallel reactions (20 ng per reaction), according to the manufacturer’s protocol.
4
Viral RNA Extraction from Ae. aegypti Mosquitoes
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All collected Ae. aegypti were pooled (n = 27). Five 2.8-mm ceramic beads (Peqlab, Erlangen, Germany) and 1 ml of Dulbecco’s phosphate-buffered saline (DPBS) were added prior to mechanical homogenization using a TissueLyser II (Qiagen, Venlo, the Netherlands). A virus purification and enrichment protocol (VIPEP) was performed on mosquito homogenates as described previously [14 (link)]. In brief, mosquito homogenizates were resuspended in DPBS pH 7 (no calcium, no magnesium; Thermo Fisher Scientific, Waltham, MA) to stabilize virus-like particles. Samples were then cleared of cells and cell debris by two centrifugation steps (5 min at 2500 g and 15 min at 4800 g) and subsequent filtration through a 0.45-µM syringe filter. Sample volumes of virus particle suspensions were reduced by ultrafiltration using 50-kDa molecular weight cut-off filtration units (Amicon Ultra-15 50 K; Merck Millipore, Cork, Ireland) before DNase and RNase treatment to eliminate contaminating nucleic acids. Viral DNA and RNA were isolated using a QIAamp UCP Micro Kit (Qiagen). Then, ribosomal RNA sequences were blocked and viral RNA was used for complementary DNA (cDNA) synthesis. Finally, viral genomic DNA and cDNA were concentrated by SpeedVac (Thermo Fisher Scientific) and amplified using a multiple displacement amplification-based Repli-g kit (Qiagen).
5
Genomic Diversity in Outcrossing Nematodes
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To study the genomic pattern of diversity in outcrossing nematodes, we sequenced 14 wild individuals of C. remanei. Isopods, a phoretic host carrier of C. remanei [193 (link)], were collected at the same station as the strains used for the genetic map, the Koffler Scientific Reserve in Ontario, Canada, in September 2013, sacrificed within a few hours following collection after having been placed on agar plates seeded with Escherichia coli OP50. From each of 14 single C. remanei individuals isolated the next day from these samples (S3 Table ), genomic DNA was directly amplified using the Repli-G kit (Qiagen), and then sequenced with Illumina HiSeq from TruSeq gDNA libraries by GenomeQuebec. One pair of C. remanei individuals derived from a shared isopod host (NS50-1, NS50-2), whereas all other individuals were isolated from different isopods.
To compare the population diversity patterns of this outcrossing species with a selfing species using similar approaches, we re-analyzed genomic sequences of 28 wild isolates of C. elegans collected at one location on the Big Island, Hawaii, from Crombie et al. [81 (link)]. For more details on the C. elegans sample, see Source data 1 from [81 (link)]. Sample IDs and the NCBI Sequence Read Archive accession numbers for C. elegans and C. remanei are inS3 Table .
To compare the population diversity patterns of this outcrossing species with a selfing species using similar approaches, we re-analyzed genomic sequences of 28 wild isolates of C. elegans collected at one location on the Big Island, Hawaii, from Crombie et al. [81 (link)]. For more details on the C. elegans sample, see Source data 1 from [81 (link)]. Sample IDs and the NCBI Sequence Read Archive accession numbers for C. elegans and C. remanei are in
6
Preparation of Methylation Controls
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After mtDNA enrichment and linearization, we prepared a negative (FU = fully unmethylated) control sample from differentiated HepaRG mtDNA by performing whole genome amplification using a repliG kit (Qiagen) according to manufacturer’s instructions. After amplification, a positive control for methylation (FM = fully methylated) was prepared. Briefly, CpG dinucleotides were methylated by incubating 1 µg of DNA with S-Adenosyl methionine (SAM) (32 µM) with CpG Methyltransferase (M.SssI) (4–25 units) (New England BioLabs) at 37 °C for 1 h before heating to 65 °C for 20mins.
7
Whole-Genome Sequencing of Tumor and Endometriosis Samples
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For WGSS, genomic DNA from frozen tumour sections or buffy coat from blood (germline reference) was extracted using the Gentra Pure Gene DNA Kit and standard techniques, as previously described. This DNA was whole‐genome amplified (WGA), using the Repli‐G kit (Qiagen), prior to use in the Haloplex amplicon kit (Agilent) for deep sequencing. DNA from additional tumours, endometriosis and benign lesions were isolated from formalin‐fixed, paraffin‐embedded (FFPE) tissues by coring, macrodissection from haematoxylin and eosin (H&E)‐stained sections, or laser‐capture microdissection (LCM) from stained sections, depending on their size and location (see supplementary material, Table S1). DNA from cored or macrodissected FFPE tissue was extracted using the QIAamp DNA FFPE kit (Qiagen). DNA from LCM FFPE tissues was extracted using the Picopure DNA Kit (Life Technologies). Samples with < 150 ng yield were subject to whole‐genome amplification, using the Ovation FFPE Whole Genome Amplification Kit (Nugen) and Ovation dsDNA module (Nugen), as marked in Table S1 (see supplementary material).
8
Whole Exome Sequencing of Tumor Relapse
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Whole exome capture was performed on DNA isolated from 48-paired diagnosis and relapse patient samples and patient-matched germline samples. Germline DNA (lymphocytes) was subjected to whole genome amplification (repli-g kit; Qiagen) in 25 patients due to limited materials (see Supplementary Table 2 ). To obtain sufficient quantities of DNA from the remaining samples, T cells were expanded ex vivo (detailed under Sample collection and processing). DNA was extracted using standard techniques. Libraries were prepared per manufacturer’s recommendation using NimbleGen SeqCap EZ Human Exome Library v3.0, Agilent Human Exon V3 (Exon 50Mb), or Agilent SureSelect Human All Exon V4 (51 MB; see Supplementary Table 2 for specification of kit use per patient tumor and germline samples) and sequenced at a minimum of 50bp single-read sequencing on a HiSeq 2000 (Illumina, Inc.) to a mean coverage per base of 73X. See Supplementary Table 4 for sequencing statistics.
9
Single-cell whole genome amplification
10
Nanopore Sequencing to Improve Achromatium Genome
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Nanopore sequencing was conducted to improve the genome recovery of Achromatium from Lake Stechlin, as an addition to the data obtained in Ionescu et al. (2017) (link). To obtain the DNA concentrations necessary for NanoPore sequencing, single Achromatium cells were obtained as previously described (Ionescu et al. 2017 (link)) and amplified using the Repli-G kit (Qiagen, Hilden, Germany) following the manufacturer instructions. Libraries for NanoPore sequencing were then prepared using the LSK-108 Kit following the manufacturer protocol, while excluding the size filtration. The prepared libraries were loaded on MIN107 R9 cells.
To incorporate the obtained reads into the previous assembly avoiding strand inversions incorporated by the phi29 polymerase in longer reads, the NanoPore reads were fragmented using the fastaslider.pl script from the Enveomics toolkit (Rodriguez-R and Konstantinidis 2016 (link)) into 250-nt-long single-end reads. These reads were then coassembled with the previous data using SPAdes assembler (Bankevich et al. 2012 (link)). However, no longer contigs than previously reported nor new functionality was obtained as a result from this assembly. The data are available at the SRA under project number PRJNA633773.
To incorporate the obtained reads into the previous assembly avoiding strand inversions incorporated by the phi29 polymerase in longer reads, the NanoPore reads were fragmented using the fastaslider.pl script from the Enveomics toolkit (Rodriguez-R and Konstantinidis 2016 (link)) into 250-nt-long single-end reads. These reads were then coassembled with the previous data using SPAdes assembler (Bankevich et al. 2012 (link)). However, no longer contigs than previously reported nor new functionality was obtained as a result from this assembly. The data are available at the SRA under project number PRJNA633773.
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