Name: amplification, next-generation sequencing, and genomic dna mapping of retroviral integration sites
Uploaded: 2016-03-22T15:00:00.000+00:00
Duration: 9 min 31 s
Description: Watch this Scientific Journal Video about Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites at JoVE.com

We are grateful to our colleagues Stephen Hughes and Henry Levin for advice that was critical to establish the NGS protocol for retroviral integration site sequencing in the Engelman lab. This work was supported by US National Institutes of Health grants AI039394 and AI052014 (to A.N.E.) and AI060354 (Harvard University Center for AIDS Research).

The authors have nothing to disclose.

A protocol for the analysis of retroviral integration sites, from the initial virus infection step through mapping of genomic distribution patterns, is described. This protocol is applicable to any retrovirus and any infectable cell type. Furthermore, the assay pipeline is quite sensitive, with the potential to recover a satisfactory number of unique integration sites from serial dilutions of genomic DNA equivalent to that of an infection initiated with an MOI of 6.4 x 10-5. This sensitivity makes the protocol...

Integration of viral DNA (vDNA) into the host cell genome is an essential step in the retroviral life cycle. Integration is accomplished by the viral enzyme integrase (IN), which carries out two distinct catalytic processes that lead to the establishment of the stably inserted provirus 1. IN subunits engage the ends of the linear vDNA that is generated through reverse transcription, forming the higher-order intasome with vDNA ends held together by an IN multimer 2-4. IN cleaves the 3&#39; ends of the vDNA downstream from invariant 5&#39;-CA-3&#39; sequences in a process known as 3&#39;-processing, leaving recessed 3&#39; ends with reactive hydroxyl groups at each vDNA terminus 5-8. The intasome is subsequently imported into the nucleus as part of a large assembly of host and viral proteins known as the preintegration complex (PIC) 9-11. After encountering cellular target DNA (tDNA), IN uses the vDNA 3&#39;-hydroxyl groups to cleave the tDNA top and bottom strands in a staggered fashion and simultaneously joins the vDNA to tDNA 5&#39; phosphate groups through the process of strand transfer 12,13.
Retroviruses exhibit integration site preferences on the local and global scales. Locally, consensus integration sites consist of weakly conserved palindromic tDNA sequences that span from approximately five to ten bp upstream and downstream from the vDNA insertion sites 14,15. Globally, retroviruses target specific chromatin annotations 16. There are seven different retroviral genera - alpha through epsilon, lenti, and spuma. The lentiviruses, which include HIV-1, favor integration within the bodies of actively transcribed genes 17, while the gammaretroviruses preferentially integrate into transcriptional start sites (TSSs) and active enhancer regions 18-20. In sharp contrast, spumavirus is strongly biased towards heterochromatic regions, such as gene-poor lamina-associated domains 21. Local tDNA base preferences are in large part dictated by specific networks of nucleoprotein contacts between IN and tDNA 13,22,23. For the lentiviruses and gammaretroviruses, integration relative to genomic annotations is in large part governed by interactions between IN and cognate cellular factors 24-27. Altering the specifics of the IN-tDNA interaction network 13,22,23,28 and disrupting or re-engineering IN-host factor interactions 25-27,29-32 are proven strategies to retarget integration on the local and global levels, respectively.
The power of DNA sequencing procedures used to catalogue retroviral integration sites has increased immensely over the past decades. Integration sites were recovered in pioneering work using laborious purification and manual cloning techniques to yield just a handful of unique sites per study 33,34. The combination of LM-PCR amplification of LTR-host DNA junctions with the ability to map individual integration sites to human and mouse draft genomes transformed the field, with the number of sites recovered from exogenous tissue culture cell infections increasing to several hundred to thousands 17,18. The more recent combination of LM-PCR with NGS methodology has sent library depth skyrocketing. Specifically, pyrosequencing yielded on the order of tens of thousands of unique integration sites 30,35-38, while libraries sequenced through the use of DNA clustering can yield millions of unique sequences 19-21,39. Here we describe an optimized LM-PCR protocol for amplifying and sequencing retroviral integration sites using DNA clustering NGS. The method incorporates required adapter sequences into the PCR primers and hence directly into the amplified DNA molecules, thereby precluding the requirement for an additional adapter ligation step prior to sequencing 40. The bioinformatic analysis pipeline, from the parsing of raw sequencing data for LTR-host DNA junctions to the mapping of unique integration sites to pertinent genomic features, is also generally described. In accordance with the precedence established from prior methodological protocols in this field 36,38,41-43, custom scripts can be developed to aid the completion of specific steps in the bioinformatics pipeline. The utility and sensitivity of the protocol is illustrated with representative data by amplifying, sequencing, and mapping HIV-1 integration sites from tissue culture cells infected at the approximate multiplicity of infection (MOI) of 1.0, as well as a titration series of this DNA diluted through uninfected cellular DNA in 5-fold steps to a maximum dilution of 1:15,625 to yield the approximate equivalent MOI of 6.4 x 10-5.

<table><tbody><tr><td>DMEM</td><td>Gibco</td><td>11965-084</td><td>Standard cell culture medium, compatible with HEK293T cells</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Scientific</td><td>SH 30088.03</td><td>Different lots of serum may need to be pre-screened for optimal viral production</td></tr><tr><td>Penicillin/Streptomycin</td><td>Corning</td><td>30-002-Cl</td><td>Antibiotics to be added to DMEM</td></tr><tr><td>Phosphate-Buffered saline</td><td>Mediatech</td><td>21-040-CV</td><td>Used to wash cells</td></tr><tr><td>Trypsin EDTA</td><td>Corning</td><td>25-053-CI</td><td>Used to detach adherent cells from tissue culture plates</td></tr><tr><td>PolyJet</td><td>SignaGen Laboratories</td><td>SL100688</td><td>DNA transfection reagent</td></tr><tr><td>0.45 &amp;micro;m Filters</td><td>Thermo Scientific</td><td>09-740-35B</td><td>Used to filter virus particle-containing cell culture media</td></tr><tr><td>Turbo DNase</td><td>Ambion</td><td>AM2239</td><td>Used to degrade carryover plasmid DNA from virus stocks</td></tr><tr><td>HIV-1 p24 Antigen Capture Assay</td><td>ABL Inc.</td><td>5447</td><td>Used to quantify yield of virus production</td></tr><tr><td>DNeasy Blood &amp;amp; Tissue Kit</td><td>Qiagen</td><td>69506</td><td>Used to purify genomic DNA from cells</td></tr><tr><td>Sonicator</td><td>Covaris</td><td>S2</td><td>With this model of sonicator perform two rounds of duty cycle, 5%; intensity, 3; cycles per burst, 200; time, 80 sec</td></tr><tr><td>Nuclease-Free Water</td><td>GeneMate</td><td>G-3250-125</td><td>Commercially-available water is recommended to reduce the possibility of sample cross-contamination</td></tr><tr><td>QIAQuick PCR Purification Kit</td><td>Qiagen</td><td>28106</td><td>Used to purify DNA during library construction</td></tr><tr><td>End-It DNA End-Repair Kit</td><td>Epicentre</td><td>ER81050</td><td>Used to repair DNA ends of sonicated DNA samples</td></tr><tr><td>Klenow Fragment (3&#039;-5&#039; exo&amp;ndash;)</td><td>New England Biolabs (NEB)</td><td>M0212S</td><td>Used with dATP to A-tail repaired DNA fragments</td></tr><tr><td>dATP</td><td>Thermo Scientific</td><td>R0141</td><td>Deoxyadenosine triphosphate</td></tr><tr><td>MseI</td><td>NEB</td><td>R0525L</td><td>Restriction endonuclease for genomic DNA cleavage</td></tr><tr><td>BglII</td><td>NEB</td><td>R0144L</td><td>Restriction endonuclease to suppress amplification of upstream HIV-1 U5 sequence</td></tr><tr><td>T4 DNA Ligase</td><td>NEB</td><td>M0202L/6218</td><td>Enzyme for covalent joining of compatible DNA ends</td></tr><tr><td>DNA Oligonucleotides</td><td>Integrated DNA Technologies</td><td>custom</td><td>Have the company purify the oligos. HPLC purification suffices for DNAs &amp;lt;30 nucleotides; PAGE purify longer DNAs&amp;nbsp;</td></tr><tr><td>Advantage 2 Polymerase Mix</td><td>Clontech</td><td>639202</td><td>Commercial mix containing DNA polymerase for PCR</td></tr><tr><td>dNTPs (100 mM solutions)</td><td>Thermo Scientific</td><td>R0181</td><td>Dilute the four chemicals on ice with sterile water to reach the intermediate worrking concentrations of 2.5 mM each dNTP</td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td>NanoDrop 2000</td><td>Spectrophotometer for determination of DNA concentration</td></tr><tr><td>Qubit Fluorimeter</td><td>Life Technologies</td><td>Qubit&amp;reg; 3.0</td><td>Fluorometer used to confirm integration site library DNA concentration</td></tr><tr><td>2200 TapeStation System</td><td>Agilent</td><td>G2964AA</td><td>Tape-based assay to confirm integration site library DNA size distribution</td></tr><tr><td>MiSeq</td><td>Illumina</td><td>SY-410-1003</td><td>Used for NGS</td></tr></tbody></table>

amplification, next-generation sequencing, and genomic dna mapping of retroviral integration sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

Table 4 lists the results of a representative experiment to illustrate the sensitivity of NGS for recovering integration sites from a culture of infected cells. Uninfected cellular DNA was utilized to serially dilute genomic DNA from an infection in which every cell on average contained one integration 40. Dilutions were prepared in steps of five to a maximal dilution of 1:15,625. Genomic DNA in the titration series was then fragmented by sonication or by diges...

Watch this Scientific Journal Video about Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites at JoVE.com

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

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Biology

17.5K Views.  Dana-Farber Cancer Institute. We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Video: Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

Genetics

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Isolation of Genomic DNA from Mouse Tails

So this is a 10 day old mouse pup and I'm going to be taking a tail biopsy and then I'm going to extract genomic DNA from its tail. So what I'm gonna do is snip a little bit of its tail off, roughly about three millimeters, and then I'll place the piece of tail in an einor tube and I'll place the einor tube on dry ice. Now, to avoid contamination from one mouse to another, I usually mark the razor blade at the part in which I made the last cut.That way for the next mouse, I'll be sure to use a clean piece of razor blade for taking the next tail biopsy. In order to distinguish between the different mice in your litter, you're gonna need a way to identify the mice. One way to do that is by punching a hole in their ears using an ear hole puncher that's shown here.Gently take the mouse by the scruff of its neck like so turn it over and pinch its tail between your fingers. Take the ear hole puncture in the other hand and make a quick snip in the mouse's ear there. See how painless that was?Okay, so now you can see I've got some pieces of tail in these einor tubes and now I'm gonna add the solutions that will help them to digest. First I'm going to add 720 microliters of STE solution, and then I'm going to add 30 microliters of protein. Ace K.So here's a look at the piece of tail that we've cut prior to digestion.Now I'm gonna place the tail pieces on a 55 degree heating block. Many protocols call for continuous motion rocking at 55 degrees, but for my purposes this won't be necessary because I'll be vortexing the tail pieces. Now I'm gonna vortex the piece of tail for two seconds.One, two. So after vortexing, here's a look at what's left inside the einor tube here, and you could see that the piece of tail has been completely dissolved and activate the proteinase K at 70 degrees Celsius for five minutes. Quench on ice for five minutes.Spin down tail pieces in a micro centrifuge for 10 minutes at full speed. In this micro centrifuge, full speed is 13, 000 RPMs. While the digestive tails are spinning down, label some einor tubes fill the einor tubes with 750 microliters of isopropanol.After spinning down the digested tails, the hair and undigested material will form a pellet at the bottom of the tube decant. The solution containing the digested material, STE and proteinase K into the einor tube containing the isopropanol DNA will begin to come out of solution, will appear ghostlike and feather like, and right there swimming around that bubble is genomic DNA that's been isolated from the mouse's tail. After mixing the isopropanol and STE solution, the genomic DNA will come out of solution and it is appear as this little ball right here, spin down the precipitated genomic DNA for five minutes at full speed in a micro centrifuge.There at the bottom of the tube is a look at our pelleted genomic DNA. Now I'm going to aspirate out the solution inside the tube, leaving only the pellet of genomic DNA behind. Each time I aspirate another tube, I'll change the pipe.Pet tip on the tip of this aspirator. After aspirating away the STE and isopropanol, I'll add one milliliter of 70%ethanol to wash the genomic DNA pellet. Because the pellet of genomic DNA usually adheres to the side of a micro fuge tube and is difficult to get off to thoroughly wash it in the 70%ethanol, you'll need to rake it across the micro fuge tube rack I have here.You can do that like so. So here's a look at the genomic DNA after it's been pelleted and washed in 70%ethanol. I'm gonna spin this down again at full speed for five minutes and then aspirate away the 70%ethanol spin down the genomic DNA pellets after they've been washed.Their 70%ethanol five minutes at full speed, going to aspirate the 70%ethanol and let the DNA dry for five minutes. There's a look at the genomic DNA pellet at the bottom of the tube, and that's about as dry as you want to get it. So I've dried out the pellets of genomic DNA from the mouse tails, and now I'm gonna add a hundred microliters of 40 millimolar triss at pH eight.So I've got the genomic DNA at 50 degrees Celsius, where it will go into solution more rapidly than add room temperature. After about an hour or so at 50 degrees, I'll either freeze it or put it at four degrees until I want to run my PCR reactions to actually figure out which mice are positive for my transgene.

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

InSET: A Novel Tool for Unbiased Genome-Wide Functional Element Discovery

Identification of Functionally-Relevant Lentivirus Integration Sites in an Insertional Mutagenesis Cell Library

The extent of functional sequences within the human genome is a pivotal yet debated topic in biology. Although high-throughput reverse genetic screens have made strides in exploring this, they often limit their scope to known genomic elements and may introduce non-specific effects. This underscores the urgent need for novel functional genomics tools that enable a deeper, unbiased understanding of genome functionality. This protocol introduces the Insertion-based Screen for functional Elements and Transcripts (InSET), a method for identifying lentivirus integration sites within a lentivirus-based insertional mutagenesis cell library. InSET facilitates the capture of genome-wide lentiviral integration sites, with next-generation sequencing used to detect and quantify flanking sequences. InSET's design enables the analysis of integration site abundance variations in phenotypic screens on a large scale, establishing it as a robust tool for forward genetics and for identifying functional genomic elements. A key benefit of InSET is its capacity to reveal previously unidentified genomic elements, including novel functional exons of both protein-coding and non-coding RNAs, independent of prior annotation. Overall, InSET holds significant value in studying the intricate complexity of the human genome and transcriptome, where many genomic elements await functional characterization.

Insertional mutagenesis is an essential tool in forward genetics for identifying functional genomic elements. Here, we describe the Insertion-based Screen for functional Elements and Transcripts (InSET), a method for detecting lentivirus integration sites within a lentivirus-based insertional mutagenesis cell library.

The extent of functional sequences within the human genome is a pivotal yet debated topic in biology. Although high-throughput reverse genetic screens ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify&#160;unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3&#8217;- and 5&#8217;- sequences adjacent to the integrated lentiviral vector.

Linear-amplification mediated (LAM)-PCR is a method developed to identify the exact positions of integrating viral vectors in the genome. The technique has evolved to be the superior method to study clonal dynamics in gene therapy patients, biosafety of novel vector technologies, T-cell diversity, cancer stem cell models, etc.

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with ...