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 border="0" cellpadding="0" cellspacing="0" width="1326">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">DMEM</td>
			<td style="width:199px;">Gibco</td>
			<td style="width:111px;">11965-084</td>
			<td style="width:780px;">Standard cell culture medium, compatible with HEK293T cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Fetal Bovine Serum</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td style="width:111px;">SH 30088.03</td>
			<td>Different lots of serum may need to be pre-screened for optimal viral production</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Penicillin/Streptomycin</td>
			<td style="width:199px;">Corning</td>
			<td style="width:111px;">30-002-Cl</td>
			<td>Antibiotics to be added to DMEM</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Phosphate-buffered saline</td>
			<td style="width:199px;">Mediatech</td>
			<td style="width:111px;">21-040-CV</td>
			<td>Used to wash cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Trypsin EDTA</td>
			<td style="width:199px;">Corning</td>
			<td style="width:111px;">25-053-CI</td>
			<td>Used to detach adherent cells from tissue culture plates</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">PolyJet</td>
			<td style="width:199px;">SignaGen Laboratories</td>
			<td style="width:111px;">SL100688</td>
			<td>DNA transfection reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:237px;">0.45 &#181;m Filters</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td>09-740-35B</td>
			<td>Used to filter virus particle-containing cell culture media</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Turbo DNase</td>
			<td style="width:199px;">Ambion</td>
			<td style="width:111px;">AM2239</td>
			<td>Used to degrade carryover plasmid DNA from virus stocks</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">HIV-1 p24 Antigen Capture Assay</td>
			<td style="width:199px;">ABL Inc.</td>
			<td style="width:111px;">5447</td>
			<td>Used to quantify yield of virus production</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">DNeasy Blood &#38; Tissue Kit</td>
			<td style="width:199px;">Qiagen</td>
			<td style="width:111px;">69506</td>
			<td>Used to purify genomic DNA from cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Sonicator</td>
			<td style="width:199px;">Covaris</td>
			<td style="width:111px;">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 height="19">
			<td height="19" style="height:19px;width:237px;">Nuclease-Free Water</td>
			<td style="width:199px;">GeneMate</td>
			<td style="width:111px;">G-3250-125</td>
			<td>Commercially-available water is recommended to reduce the possibility of sample cross-contamination</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">QIAQuick PCR Purification Kit</td>
			<td style="width:199px;">Qiagen</td>
			<td style="width:111px;">28106</td>
			<td>Used to purify DNA during library construction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">End-It DNA End-Repair Kit</td>
			<td style="width:199px;">Epicentre</td>
			<td style="width:111px;">ER81050</td>
			<td>Used to repair DNA ends of sonicated DNA samples</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Klenow Fragment (3&#39;-5&#39; exo&#8211;)</td>
			<td style="width:199px;">New England Biolabs (NEB)</td>
			<td style="width:111px;">M0212S</td>
			<td>Used with dATP to A-tail repaired DNA fragments</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">dATP</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td style="width:111px;">R0141</td>
			<td>Deoxyadenosine triphosphate</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">MseI</td>
			<td style="width:199px;">NEB</td>
			<td>R0525L</td>
			<td>Restriction endonuclease for genomic DNA cleavage</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">BglII</td>
			<td style="width:199px;">NEB</td>
			<td style="width:111px;">R0144L</td>
			<td>Restriction endonuclease to suppress amplification of upstream HIV-1 U5 sequence</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">T4 DNA Ligase</td>
			<td style="width:199px;">NEB</td>
			<td style="width:111px;">M0202L/6218</td>
			<td>Enzyme for covalent joining of compatible DNA ends</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DNA Oligonucleotides</td>
			<td>Integrated DNA Technologies</td>
			<td>custom</td>
			<td>Have the company purify the oligos. HPLC purification suffices for DNAs &#60;30 nucleotides; PAGE purify longer DNAs&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Advantage 2 Polymerase Mix</td>
			<td style="width:199px;">Clontech</td>
			<td style="width:111px;">639202</td>
			<td>Commercial mix containing DNA polymerase for PCR</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">dNTPs (100 mM solutions)</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td style="width:111px;">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 height="19">
			<td height="19" style="height:19px;width:237px;">NanoDrop</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td style="width:111px;">NanoDrop 2000</td>
			<td>Spectrophotometer for determination of DNA concentration</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Qubit Fluorimeter</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:111px;">Qubit&#174; 3.0</td>
			<td>Fluorometer used to confirm integration site library DNA concentration</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">2200 TapeStation System</td>
			<td style="width:199px;">Agilent</td>
			<td style="width:111px;">G2964AA</td>
			<td>Tape-based assay to confirm integration site library DNA size distribution</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">MiSeq</td>
			<td style="width:199px;">Illumina</td>
			<td style="width:111px;">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 ...

amplification-next-generation-sequencing-genomic-dna-mapping

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JoVE Journal

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

Isolation of Genomic DNA from Mouse Tails

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 ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...