אנו מודים עמיתינו סטיבן יוז והנרי לוין לייעוץ היה קריטי להקים פרוטוקול NGS עבור סידור האתר אינטגרציה retroviral במעבדה אנגלמן. עבודה זו נתמכה על ידי ארצות הברית המכונים הלאומיים לבריאות מענקים AI039394 ו AI052014 (כדי Ane) ו AI060354 (מרכז אוניברסיטת הרווארד לחקר האיידס).

The authors have nothing to disclose.

פרוטוקול לניתוח אתרי אינטגרציה retroviral, משלב הזיהום בנגיף הראשוני ועד מיפוי של דפוסי תפוצה גנומית, מתואר. פרוטוקול זה חל על כל רטרו-וירוס וכל סוג תא infectable. יתר על כן, צינור assay רגישה למדי, עם פוטנציאל להתאושש מספר משביע רצון של אתרי שילוב ייחודי בין דילולים סדרתי של שווה ?...

שילוב של ה- DNA הנגיפי (vDNA) לתוך הגנום של התא הפונדקאי הוא צעד חיוני במחזור חי retroviral. אינטגרציה מושגת על ידי integrase אנזים ויראלי (IN), אשר מבצע שני תהליכים קטליטיים ברורים שיובילו להקמת provirus מוכנס ביציבות 1. לפי יחידות משנה להעסיק את הקצוות של vDNA ליניארי כי נוצר באמצעות שעתוק לאחור, להרכיב את intasome מהסדר הגבוה עם vDNA מסתיים מחוזקת ביחד בידי לפי multimer 2-4. לפי וחותך את &#39;הקצוות של vDNA במורד הזרם 5&#39;-CA-3 משתנה&#39; 3 רצפים תהליך המכונה 3&#39;-עיבוד, בהותירה את הפסקה 3 &#39;מסתיים עם קבוצות הידרוקסיל תגובתי בכל vDNA הסופית 5-8. Intasome מיובא לאחר מכן לתוך הגרעין במסגרת אסיפה גדולה של מארח חלבונים נגיפיים הידועים כמתחם preintegration (PIC) 9-11. לאחר המפגש עם המטרה תאית DNA (tDNA), לפי משתמשת vDNA 3&#39;-הידרוקסיל Groעליות לדבוק העליון tDNA וקצובות תחתונות בצורה מדורגת ובמקביל מצטרף vDNA לקבוצות פוספט &#39;tDNA 5 בתהליך של גדיל העברה 12,13. העדפות אינטגרציה באתר התערוכה Retroviruses על המאזניים המקומיים והעולמיים. מקומית, אתרי אינטגרצית קונסנסוס מורכבים רצפי tDNA palindromic נשמרים בחולשה כי ההיקף בערך מחמישה עשר נ&quot;ב במורד זרם מאתרי כניסת vDNA 14,15. גלובלי, רטרווירוסים למקד הסברים ספציפיים הכרומטין 16. ישנם שבעה סוגים retroviral שונים - אלפא באמצעות אפסילון, lenti, ו spuma. Lentiviruses, הכולל HIV-1, לטובת השתלבות גופות הגנים לרנ&quot;א 17, בעוד gammaretroviruses לשלב מועדף למתחמי תחילת תעתיק (TSSs) ואזורים משפרים פעילים 18-20. בניגוד חד, spumavirus מוטה חזק כלפי heterochromאזורי atic, כגון 21 תחומי גן-עני הקשורים-lamina. העדפות בסיס tDNA מקומיות הם חלק גדול מוכתב על ידי רשתות ספציפיות של אנשי קשר nucleoprotein שבין פנימי tDNA 13,22,23. עבור lentiviruses ו gammaretroviruses, ביחס אינטגרציה ל- ביאורים הגנומי הוא במידה רבה מוסדר על ידי אינטראקציות בין לפי וגורמים הסלולר מאותו מקור 24-27. שינוי הפרטים של רשת אינטראקציה IN-tDNA 13,22,23,28 ושיבוש או Re-engineering IN-מארח אינטראקציות גורם 25-27,29-32 מוכחות אסטרטגיות כדי להתמקד מחדש אינטגרציה במישור המקומי והעולמי, בהתאמה. כוחה של נהלי רצפי DNA היה מקטלגי אתרי אינטגרצית retroviral בכדור הארץ גבר מאוד בעשורים האחרונים. אתרי אינטגרציה שנמצאו בעבודתו החלוצית באמצעות טיהור מייגעת וטכניקות שיבוט ידניות להניב רק קומץ של אתרים ייחודיים לכל 33,34 מחקר.השילוב של הגברת LM-PCR של צמתי DNA LTR-מארח עם היכולת למפות אתרי אינטגרצית פרט הגנום אנושי ועכבר טיוטה הפך את השדה, עם מספר האתרים התאוששו מזיהומי תרבית תאי אקסוגניים רקמות הגדיל לכמה מאה לאלפי 17 , 18. השילוב עדכנית יותר של LM-PCR עם מתודולוגיה NGS שלח נסיקת עומק הספרייה. באופן ספציפי, pyrosequencing הניב בסדר גודל של עשרות אלפי אתרים שילוב ייחודי 30,35-38, בעוד ספריות רצף באמצעות אשכולות DNA יכול להניב מיליוני רצפים ייחודיים 19-21,39. כאן אנו מתארים פרוטוקול LM-PCR אופטימיזציה עבור הגברה וסדר אתרי אינטגרציה retroviral באמצעות NGS אשכולות DNA. השיטה משלבת נדרשת רצפי מתאם לתוך פריימרים PCR ומכאן ישירות לתוך מולקולות דנ&quot;א המוגברות, ובכך מניעת דרישת צעד קשירת מתאם נוסף לפני sequencing 40. צינור ניתוח bioinformatic, מן הניתוח של נתוני רצף גלם צומת DNA LTR-לארח את המיפוי של אתרי אינטגרציה ייחודיים רלוונטי תכונות גנומית, גם מתואר בדרך כלל. בהתאם עדיפות הוקמה מתוך פרוטוקולים מתודולוגיים לפני בתחום זה 36,38,41-43, סקריפטים מותאמים אישית ניתן לפתח על מנת לסייע בהשלמת צעדים ספציפיים בצנרת ביואינפורמטיקה. השירות והרגיש של הפרוטוקול מודגם עם נתונים נציג ידי הגברה, רצף, ומיפוי אתרי אינטגרציה HIV-1 מתאי בתרבית רקמה הנגוע על הריבוי המשוער של זיהום (משרד פנים) של 1.0, כמו גם סדרת טיטרציה של DNA זה מדולל באמצעות הדנ&quot;א תאי נגוע ב 5-לקפל צעדים לדילול מקסימלית של 1: 15,625 להניב משרד הפנים המקבילים המשוער של 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

תערוכת Retroviruses העדפות אינטגרצית חתימה על המאזניים המקומיים ועולמיים כאחד. כאן, אנו מציגים פרוטוקול מפורט (1) דור של ספריות מגוונות של אתרי אינטגרציה retroviral באמצעות בתיווך קשירת PCR (LM-PCR) הגברת רצף של הדור הבא (NGS), (2) מיפוי המיקום הגנומי של כל virus- לארח צומת באמצעות BEDTools, ו (3) ניתוח הנתונים עבור הרלוונטיות סטטיסטיות. הדנ&quot;א הגנומי מופק בתאים נגועים מפוצל על ידי עיכול עם אנזימי הגבלה או על ידי sonication. לאחר תיקון סוף DNA המתאים, linkers פעמים התקועות הוא ligated על DNA מסתיים, חצי מקונני PCR מתבצעת באמצעות פריימרים משלימים הוא חוזר מסוף הארוך (LTR) בסופו של הנגיף לבין DNA מקשר ligated. פריימרים PCR לבצע רצפים הנדרשים אשכולות DNA במהלך NGS, השוללים את דרישת קשירת מתאם נפרדת. בקרת איכות (QC) נערכה על מנת להעריך התפלגות גודל שבר דנ&quot;א ולהתאיםאה DNA התאגדות לפני NGS. קבצי פלט רצף מסוננים עבור LTR המכיל קורא, ואת הרצפים בהגדרת LTR והמקשר נחתכים משם. רצפים התא המארח גזוז ממופים הגנום התייחסות באמצעות בלאט ו מסוננים עבור זהות מינימלית 97% עד לנקודה ייחודית בגנום התייחסות. אתרי אינטגרציה ייחודיים בחן עבור נוקלאוטיד הסמוך (NT) ביחס רצף והפצה לתכונות גנומי שונות. שימוש בפרוטוקול זה, ספריות באתר אינטגרציה גבוהות של מורכבות ניתן לבנות הדנ&quot;א הגנומי בשלושה ימים. הפרוטוקול כולו שמקיף זיהום ויראלי אקסוגני של תאים בתרבית רקמה ניתן לאנליזה האתר אינטגרציה ולכן יכול להתנהל כ שבוע עד שבועיים. היישומים אחרונים של טכנולוגיה זו נוגעים ניתוח אורך של אתרי אינטגרציה מחולים עם HIV.

רשימות לוח 4 תוצאות ניסוי נציג כדי להמחיש את הרגישות של NGS לשחזור אתרי אינטגרציה מתרבה של תאים נגועים. DNA התאי הנגוע נוצל כדי לדלל הדנ&quot;א הגנומי סדר מזיהום שבו כל תא בממוצע הכיל שילוב אחד 40. דילולים נערכו שלבים של חמש עד דילול מקסימאלי ...

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.

הגברה, רצף הדור הבא, ואת הגנום מיפוי ה- DNA של אתרי אינטגרצית retroviral

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

Research

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

בידוד של הדנ&quot;א הגנומי של זנבות עכבר

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.

הגנום MRI - משאב ציבורי עבור לימוד דפוסי בתוך רצף הדנ&quot;א הגנומי

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.

מסכי הגנום תחרותי של ספריות שמרים המבורקדים

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.

פלואורסצנטי, מיקרוסקופ הקרנת ואת הדור הבא הרצף: כלים שימושיים לזיהוי גנים המעורבים אברון Integrity

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.

לכידה סלקטיבית של 5-hydroxymethylcytosine מהדנ&quot;א הגנומי

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.

רצף של הדור הבא של 16S ריבוזומלי amplicons ג&#39;ין RNA

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.

ממוקד DNA מתילציה ניתוח על ידי רצף של הדור הבא

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.

DamID-seq: מיפוי הגנום של אינטראקציות החלבון-DNA על ידי תפוקה גבוהה רצף של קטעי DNA-מפוגל אדנין

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.

ממוקד RNA רצף Assay לאפיון ביטוי גנים ושינויים הגנום

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.

Interactome-Seq: פרוטוקול עבור ספריית Domainome ובניה, אימות בחירה Phage התצוגה ורצף הדור הבא

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