This work was supported by grant NIH GM117446 to A.B.

1. Preparation of Cells for Flow Cytometry Sorting
<ol>
	<li>Inoculate 15 mL test tubes containing 8 mL each of YEPD broth such that the cultures reach a density of 5 x 106 to 1.5 x 107 cells per mL after overnight growth (see discussion note 1).</li>
	<li>Spin down yeast cells (1,400 x g, at room temperature or 4 &#176;C) in a 15 mL screw cap centrifuge tube for 5 min, resuspend cells in 1.5 mL 70% ethanol, and transfer to a 1.6 mL microfuge tube, letting this sit for 1 h at room temperature or at least 3 h on ice (can be stored indefinitely at 4 &#176;C) (see Discussion point (2)).</li>
	<li>Spin down yeast cells in the microfuge (14,000 x g, 4 &#176;C) for 1 min, resuspend in 1 mL 50 mM sodium citrate, pH 7.2, spin down (14,000 x g, 4 &#176;C) for 1 min, aspirate supernatant, resuspend in 1 mL 50 mM sodium citrate, pH 7.2, containing 0.25 mg/mL RNAse solution for 1 h at 50 &#176;C or overnight at 37 &#176;C in heat block or water bath.</li>
	<li>Add 50 &#181;L proteinase K solution (20 mg/mL resuspended in 10 mM Tris pH 7.5, 2 mM CaCl2, 50% weight to volume glycerol) and incubate for 1 h at 50 &#176;C.</li>
	<li>Spin down cells (14,000 x g, 4 &#176;C), resuspend cells in 1 mL 50 mM sodium citrate, pH 7.2, spin down (14,000 x g, 4 &#176;C), aspirate supernatant with vacuum, resuspend in 1 mL 1 &#181;M Green nucleic acid strain resuspended in 50 mM sodium citrate, pH 7.2 and incubate in the dark for 1 h at room temperature, sonicate 2x for 2 s each (output power 2 watts).</li>
</ol>
2. Cell Sorting
<ol>
	<li>Using a cell sorter, sort cells according to DNA content into phases G1, S, &#34;early G2,&#34; and &#34;late G2&#34;, collecting at least 1.6 million haploid cells from each cell cycle phase as in Figure 1 (see Discussion point (3)).</li>
	<li>Transfer cells to microfuge tubes and spin down cells for 20 min at 14,000 x g at 4 &#176;C in the microfuge and aspirate supernatant with vacuum (the pellet can be stored frozen at -20 &#176;C indefinitely; see discussion point (4)).</li>
</ol>
3. DNA Extraction and the Preparation of Sequencing Libraries
NOTE: The following steps are based on &#34;protocol I&#34; in the Yeast Genomic DNA extraction Kit (see Table of Materials).
<ol>
	<li>Add 120 &#181;L of &#34;Digestion Buffer,&#34;&#160;a proprietary buffer that allows yeast lytic enzyme to digest the cell wall, and 5 &#181;L of yeast lytic enzyme, resuspend by vortexing, and incubate at 37 &#176;C for 40 - 60 min.</li>
	<li>Add 120 &#181;L of &#34;Lysis Buffer,&#34;&#160;a proprietary buffer that contains detergent, and vortex hard for 10 - 20 s.</li>
	<li>Add 250 &#181;L chloroform - mix thoroughly for 1 min.</li>
	<li>Spin at maximum speed in a microfuge for 2 min.</li>
	<li>Load the supernatant onto the DNA binding column and centrifuge at maximum speed in a microfuge for 1 min.</li>
	<li>Add 300 &#181;L of &#34;DNA Washing Buffer,&#34;&#160;a proprietary buffer that contains ethanol, and centrifuge for 1 min at maximum speed in a microfuge. Discard the flow through, add 300 &#181;L of DNA Wash Buffer, and repeat the wash process.</li>
	<li>Transfer the spin column to a fresh microfuge tube, add 60 &#181;L of water (not TE) - wait 1 - 3 min and then centrifuge for 10 s to elute the DNA.</li>
	<li>Measure concentration by adding 5 &#181;L of DNA to 195 &#181;L of dsDNA high sensitivity reagent that has been diluted 1:200 in dsDNA high sensitivity buffer and then measure fluorescence in a fluorometer, using 10 &#181;L of supplied standards for calibration; expect between 10 and 50 ng of DNA total yield.</li>
	<li>Sonicate (Peak Incident Power 450, Duty Factor 30%, Cycles per Burst 200, Treatment Time 60 s, Water Level 6) to break up DNA into 250 - 350 bp fragments.</li>
	<li>Prepare a library using DNA sample preparation kit and sequence it (at least 10 million 50 bp single-end reads) on the DNA sequencing instrument in rapid mode (see discussion note (5)).</li>
</ol>
4. Analysis of Sequencing Data and Generation of Replication Profiles
<ol>
	<li>Align sequences to Saccharomyces cerevisiae sacCer3 genome using Gsnap12 (see discussion note (6)).</li>
	<li>Determine per-base pair read depths using Bedtools&#39; &#34;genomeCoverageBed&#34; software13.</li>
	<li>Remove artefactual spikes in read depths, if present (see discussion note (7)).</li>
	<li>Smooth read depths by chromosome using medians in a 20 kb sliding window using &#34;runMean&#34; function in R package &#34;caTools&#34; (see discussion note (8)).</li>
	<li>Plot read depths in S phase, early G2, and late G2 as a percentage of completely replicated read depths (see discussion note (9)).</li>
</ol>

<ol>
	<li>Fragkos, M., Ganier, O., Coulombe, P., Mechali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+origin+activation+in+space+and+time.">DNA replication origin activation in space and time.</a> Nat Rev Mol Cell Biol. 16, 360-374 (2015).</li><li>Santocanale, C., Diffley, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mec1-+and+Rad53-dependent+checkpoint+controls+late-firing+origins+of+DNA+replication.">A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication.</a> Nature. 395, 615-618 (1998).</li><li>Brewer, B. J., Fangman, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+replication+fork+barrier+at+the+3'+end+of+yeast+ribosomal+RNA+genes.">A replication fork barrier at the 3' end of yeast ribosomal RNA genes.</a> Cell. 55, 637-643 (1988).</li><li>Raghuraman, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+dynamics+of+the+yeast+genome.">Replication dynamics of the yeast genome.</a> Science. 294, 115-121 (2001).</li><li>Muller, C. A., Nieduszynski, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+replication+timing+reveals+global+and+local+regulation+of+replication+origin+activity.">Conservation of replication timing reveals global and local regulation of replication origin activity.</a> Genome Res. 22, 1953-1962 (2012).</li><li>Koren, A., Soifer, I., Barkai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRC1-dependent+scaling+of+the+budding+yeast+DNA+replication+timing+program.">MRC1-dependent scaling of the budding yeast DNA replication timing program.</a> Genome Res. 20, 781-790 (2010).</li><li>Lang, G. I., Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+rates+across+budding+yeast+chromosome+VI+are+correlated+with+replication+timing.">Mutation rates across budding yeast chromosome VI are correlated with replication timing.</a> Genome Biol Evol. 3, 799-811 (2011).</li><li>Durkin, S. G., Glover, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+fragile+sites.">Chromosome fragile sites.</a> Annu Rev Genet. 41, 169-192 (2007).</li><li>Dillon, L. W., Burrow, A. A., Wang, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+instability+at+chromosomal+fragile+sites+in+cancer.">DNA instability at chromosomal fragile sites in cancer.</a> Curr Genomics. 11, 326-337 (2010).</li><li>Widrow, R. J., Hansen, R. S., Kawame, H., Gartler, S. M., Laird, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Very+late+DNA+replication+in+the+human+cell+cycle.">Very late DNA replication in the human cell cycle.</a> Proc Natl Acad Sci U S A. 95, 11246-11250 (1998).</li><li>Foss, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIR2+suppresses+replication+gaps+and+genome+instability+by+balancing+replication+between+repetitive+and+unique+sequences.">SIR2 suppresses replication gaps and genome instability by balancing replication between repetitive and unique sequences.</a> Proc Natl Acad Sci U S A. 114, 552-557 (2017).</li><li>Wu, T. D., Reeder, J., Lawrence, M., Becker, G., Brauer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GMAP+and+GSNAP+for+Genomic+Sequence+Alignment:+Enhancements+to+Speed,+Accuracy,+and+Functionality.">GMAP and GSNAP for Genomic Sequence Alignment: Enhancements to Speed, Accuracy, and Functionality.</a> Methods Mol Biol. 1418, 283-334 (2016).</li><li>Quinlan, A. R., Hall, I. 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W., Lander, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proof+and+evolutionary+analysis+of+ancient+genome+duplication+in+the+yeast+Saccharomyces+cerevisiae.">Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae.</a> Nature. 428, 617-624 (2004).</li><li>Goodwin, S., McPherson, J. D., McCombie, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coming+of+age:+ten+years+of+next-generation+sequencing+technologies.">Coming of age: ten years of next-generation sequencing technologies.</a> Nat Rev Genet. 17, 333-351 (2016).</li></ol>

The authors have nothing to disclose.

While this technique is robust and relatively straight forward, particular attention should be devoted to the following:
(1) We recommend that cultures grow for at least 12 h before they reach log phase, since differences manifest in cell cycle distributions if cultures are harvested after having reached the desired density just 4 h after inoculation. Our assumption is that a cell cycle distribution that has reached a relatively stable equilibrium better represents a &#34;real&#34; log phase d...

Eukaryotic genome replication is initiated at multiple discrete sites, called origins of replication, from which replication forks proceed in both directions (reviewed in Fragkos et al., 20151). Origins vary in both their timing and efficiency of firing, and several techniques have been developed to monitor replication origin activity and elucidate the causes of this variation. The activity of individual origins can be inferred from levels of single-stranded DNA2, which forms around active origins, or by using 2D gel electrophoresis to monitor specific replication intermediates3, both of which can be detected with radioactive probes. Both of these techniques are more easily applied in S. cerevisiae than in mammalian cells, because origins are limited to specific known sequences in the former. With the advent of microarrays, it became possible to assess origin firing globally. This was first done by labeling DNA with heavy isotopes, releasing cells from a G1 block into medium containing light isotopes, and then monitoring the formation of heavy-light hybrid DNA across the genome4. The introduction of high throughput sequencing allowed similar genome-wide monitoring of origin activity without the requirement of expensive isotopic labeling. Cells were sorted in a flow cytometer according to DNA content and their DNA subjected to deep sequencing. Because sequence coverage proceeds from 1x to 2x over the course of S phase, relative replication timing can be assessed by comparing read depths of cells in S phase to those in G1 or G25,6. These techniques, particularly applied to yeast, led to a deeper understanding of how DNA sequence, chromatin structure, and DNA replication proteins regulate origin timing and efficiency.
Faithful transmission of genetic information during cellular proliferation requires not only successful initiation of DNA replication, which takes place at origins, but also successful completion of replication, which occurs where replication forks meet. Like initiation of replication, the timing of completion of replication varies across the genome with certain regions remaining unreplicated even late in the cell cycle. Such regions may be particularly distant from active replication origins or may contain sequences or chromatin structures that impede DNA polymerases. Late replicating regions can manifest themselves as fragile sites, which are associated with chromosomal breakage and higher mutation rates, and have been implicated in cancer and aging7,8,9. However, despite the importance of proper completion of DNA replication in maintenance of genome stability, our understanding of where and how this takes place has lagged far behind that of replication initiation. And while individual genes whose late replication has been associated with disease have been studied with, for example, qPCR10, global studies directed at elucidating the locations and underlying causes of late replication have been lacking. Here we describe a technique we refer to as &#34;G2 seq&#34; in which we combine flow cytometry with high throughput sequencing to shed light on the completion of genome replication in yeast11. With minor changes, this protocol can be adapted to any cells that can be flow-sorted according to DNA content.

<table><tbody><tr><td>YeaStar Genomic DNA kit</td><td>Genesee Scientific</td><td>11-323</td><td></td></tr><tr><td>1 &amp;micro;M SYTOX Green</td><td>ThermoFisher</td><td>57020</td><td>resuspend in 50 mM sodium citrate, pH 7.2</td></tr><tr><td>50 mM sodium citrate, pH 7.2</td><td></td><td></td><td></td></tr><tr><td>RNase solution (0.25 mg / mL)</td><td>Sigma</td><td>R6513</td><td>0.25 mg / mL RNaseA resuspended in 50 mM sodium citrate, pH 7.2, boil RNase solution for 10 minutes before the first use only, and from then on store at -20&amp;deg;</td></tr><tr><td>proteinase K solution (20 mg / mL)</td><td>ThermoFisher / Invitrogen</td><td>25530-015</td><td>resuspend in 10 mM Tris, pH 7.5, 2 mM CaCl2, 50% glycerol, store at -20&amp;deg;C</td></tr><tr><td>Model 50 Sonic Dismembrator</td><td>Fisher Scientific</td><td>FB50A220</td><td></td></tr><tr><td>BD Biosciences FACSAria II</td><td>BD Biosciences</td><td>644832</td><td></td></tr><tr><td>Zymo-spin III columns</td><td>Zymo Research</td><td>C1005</td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>ThermoFisher</td><td>Q32851</td><td></td></tr><tr><td>Qubit 3.0 Fluorometer</td><td>ThermoFisher</td><td>Q33216</td><td></td></tr><tr><td>Covaris Model LE220 Focused-Ultrasonicator</td><td>Covaris</td><td>500219</td><td></td></tr><tr><td>Illumina TruSeq DNA LT Sample Prep Kit</td><td>Illumina</td><td>15026486</td><td></td></tr><tr><td>Illumina HiSeq 2500 instrument</td><td>Illumina</td><td>SY&amp;ndash;401&amp;ndash;2501</td><td></td></tr><tr><td>gsnap alignment software</td><td>open source software / Genentech</td><td></td><td>http://research-pub.gene.com/gmap</td></tr></tbody></table>

g2-seq: a high throughput sequencing-based technique for identifying late replicating regions of the genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We have used the procedure described above to identify late replicating sites in budding yeast. Testing this approach using a known late replicating region on an artificial chromosome proved the technique to be accurate and reliable. Our results have also demonstrated the biological importance of timely completion of replication by showing that a late replicating region on chromosome 7, which we identified as late replicating on the basis of our G2-seq results, was lost approximately thre...

Watch this Scientific Journal Video about G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome at JoVE.com

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

g2-seq-high-throughput-sequencing-based-technique-for-identifying

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

5.7K Views.  Fred Hutchinson Cancer Research Center. We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

Video: G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

2D Gel Electrophoresis for Analyzing DNA Replication and Fork Stalling Events

Electrophoretic Analysis of Replication Through Structure-Prone DNA Repeats Within the SV40-Based Human Episome

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This protocol describes how to analyze replication fork progression through structure-prone, expandable DNA repeats within the simian virus 40 (SV40)-based episome in human cells. In brief, upon plasmid transfection into human cells, replication intermediates are isolated by the modified Hirt protocol and treated with the DpnI restriction enzyme to remove non-replicated DNA. Intermediates are then digested by appropriate restriction enzymes to place the repeat of interest within the origin-distal half of a 3-5 kb-long DNA fragment. The replication intermediates are separated into two perpendicular dimensions, first by size and then by shape. Following Southern blot hybridization, this approach allows researchers to observe fork stalling at various structure-forming repeats on the descending half of the replication Y-arc. Furthermore, this positioning of the stall site allows the visualization of various outcomes of repeat-mediated fork stalling, such as fork reversal, the advent of a converging fork, and recombinational fork restart.

Author Spotlight: Characterizing DNA Replication of Pathogenic Repeats to Uncover Mechanisms of Replication Fork Stalling and Expansion

Here, we outline the procedure for analyzing replication progression through pathogenic, structure-prone repeats using 2-dimensional gel electrophoresis.

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...

Bioengineering

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

Medicine

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes and analysis of complex structural variants. Nanopore platforms perform single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores and can generate hundreds of kilobase (kb) reads with minimal capital cost. This platform has been adopted by many researchers for a variety of applications. Achieving longer sequencing read lengths is the most critical factor to leverage the value of nanopore sequencing platforms. To generate ultra-long reads, special consideration is required to avoid DNA breakages and gain efficiency to generate productive sequencing templates. Here, we provide the detailed protocol of ultra-long DNA sequencing including high molecular weight (HMW) DNA extraction from fresh or frozen cells, library construction by mechanical shearing or transposase fragmentation, and sequencing on a nanopore device. From 20-25 &#181;g of HMW DNA, the method can achieve N50 read length of 50-70 kb with mechanical shearing and N50 of 90-100 kb read length with transposase mediated fragmentation. The protocol can be applied to DNA extracted from mammalian cells to perform whole genome sequencing for the detection of structural variants and genome assembly. Additional improvements on the DNA extraction and enzymatic reactions will further increase the read length and expand its utility.

Long-read sequences greatly facilitate the assembly of complex genomes and characterization of structural variation. We describe a method to generate ultra-long sequences by nanopore-based sequencing platforms. The approach adopts an optimized DNA extraction followed by modified library preparations to generate hundreds of kilobase reads with moderate coverage from human cells.

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Biology

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

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Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Massively Parallel Reporter Assays in Cultured Mammalian Cells

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair