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><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

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

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.

דו כיווני Retroviral אינטגרציה אתר PCR מתודולוגיה וניתוח נתונים כמותיים זרימת עבודה

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.

Retroviral סריקה: מיפוי MLV אתרי אינטגרציה להגדרת אזורים רגולטוריים ספציפיים לתא

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.

בידוד של הדנ&quot;א הגנומי של זנבות עכבר

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.

הגברה של יד באורך מלא הפרו-וירוס HIV-1 עבור הדור הבא רצפי

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

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell-based, high-coverage, and high-resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3'-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting of whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size fractionation by gel purification, PCR amplification, deep sequencing, and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3'-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3'-extension, provided that the template sequence is known.

Here we present a deep sequencing approach that provides an unbiased determination of nascent 3&#39;-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription.

3'-Termini הקובע ורצפים של מולקולות Nascent חד גדילי ה-DNA הנגיפי במהלך HIV-1 הפוך שעתוק בתאים נגועים

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

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 ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

מדידה<em&gt; במבחנה</em&gt; פעילות האינטגרציה של HIV-1 Preintegration מכלולים

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

Immunology and Infection

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 ...

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.

ליניארי ההגברה המתווכת PCR - לוקליזציה של אלמנטים גנטיים ואפיון ידוע האיגוף DNA

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

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

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