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For more information, log on to- shomusbiology weebly com Download the study materials here- shomusbiology weebly com bio-materials html In genetics, shotgun sequencing, also known as shotgun cloning, is a method used for sequencing long DNA strands It is named by analogy with the rapidly expanding, quasi-random firing pattern of a shotgun Since the chain termination method of DNA sequencing can only be used for fairly short strands (100 to 1000 basepairs), longer sequences must be subdivided into smaller fragments, and subsequently re-assembled to give the overall sequence Two principal methods are used for this chromosome walking, which progresses through the entire strand, piece by piece, and shotgun sequencing, which is a faster but more complex process, and uses random fragments In shotgun sequencing,[1][2] DNA is broken up randomly into numerous small segments, which are sequenced using the chain termination method to obtain reads Multiple overlapping reads for the target DNA are obtained by performing several rounds of this fragmentation and sequencing Computer programs then use the overlapping ends of different reads to assemble them into a continuous sequence [1] Shotgun sequencing was one of the precursor technologies that was responsible for enabling full genome sequencing In this extremely simplified example, none of the reads cover the full length of the original sequence, but the four reads can be assembled into the original sequence using the overlap of their ends to align and order them In reality, this process uses enormous amounts of information that are rife with ambiguities and sequencing errors Assembly of complex genomes is additionally complicated by the great abundance of repetitive sequence, meaning similar short reads could come from completely different parts of the sequence Many overlapping reads for each segment of the original DNA are necessary to overcome these difficulties and accurately assemble the sequence For example, to complete the Human Genome Project, most of the human genome was sequenced at 12X or greater coverage; that is, each base in the final sequence was present, on average, in 12 reads Even so, current methods have failed to isolate or assemble reliable sequence for approximately 1% of the (euchromatic) human genome Source of the article published in description is Wikipedia I am sharing their material Copyright by original content developers Link- en wikipedia org wiki Main_Page

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Shotgun sequencing method explained

This lecture explains about the shotgun sequencing method This DNA sequencing method is used to clone and sequence large genome content during the human genome project Shotgun DNA sequencing is the best approach to sequence big DNA content after the mechanical shearing The fragmented DNA is then sequenced with Sanger method or dideoxy method or any other next generation sequencing methods of the DNA For more information, log on to- www shomusbiology com Get Shomu s Biology DVD set here- www shomusbiology com dvd-store Download the study materials here- shomusbiology com bio-materials html Remember Shomu’s Biology is created to spread the knowledge of life science and biology by sharing all this free biology lectures video and animation presented by Suman Bhattacharjee in YouTube All these tutorials are brought to you for free Please subscribe to our channel so that we can grow together You can check for any of the following services from Shomu’s Biology- Buy Shomu’s Biology lecture DVD set- www shomusbiology com dvd-store Shomu’s Biology assignment services – www shomusbiology com assignment -help Join Online coaching for CSIR NET exam – www shomusbiology com net-coaching We are social Find us on different sites here- Our Website – www shomusbiology com Facebook page- s www facebook com ShomusBiology Twitter - s twitter com shomusbiology SlideShare- www slideshare net shomusbiology Google plus- s plus google com 113648584982732129198 LinkedIn - s www linkedin com in suman-bhattacharjee-2a051661 Youtube- s www youtube com user TheFunsuman Thank you for watching the video lecture on the shotgun sequencing of the

Next Generation Sequencing Library Preparation Seq It Out #10

Submit your question at www thermofisher com ask What do you call a collection of millions of DNA fragments sharing the same short sequences on the 5’ and 3’ ends? The answer is a next generation sequencing, or NGS, library Today, we are going to focus on the four basic steps of NGS library preparation that can be broadly applied across different preparation methods A key step in the NGS workflow is preparing the input for sequencing, known as creating a library An NGS library is a collection of similarly sized DNA fragments with known adapter sequences added to the 5’ and 3’ ends A library corresponds to a single sample and multiple libraries, each with their own unique adapter sequences, can be pooled and sequenced in the same sequencing run NGS library preparation has four general steps 1 DNA Fragmentation or Target Selection, 2 Addition of adapter sequences, 3 Size selection, and 4 Final library quantification and QC Let’s take a look at our lab book The first step, DNA fragmentation or Target Selection In order to get the starting DNA into smaller pieces, isolated DNA may be fragmented using physical or enzymatic methods These libraries are referred to as fragment libraries Alternatively, if the sequence of specific DNA targets is known, PCR amplification of those targets may be used to produce DNA amplicons within the desired size range These libraries are referred to as amplicon libraries Next, specific DNA adapter sequences are annealed to the 5’ and 3’ ends of the fragmented or amplicon DNA The double-stranded DNA adapters are approximately 20 to 40bp fragments that contain known sequences Generally, there are two different adapter sequences that can anneal to the DNA fragments in either the 5’ or 3’ orientation One adapter sequence contains the primer annealing site for the sequencing primer, while the second adapter sequence is generally used to anchor the DNA fragment to a surface for sequencing; for example beads or a solid surface containing a complimentary DNA sequence Now we have our DNA fragments with known adapter sequences on either end The next step is to select the library fragment sizes we need for our sequencing run There are two common size selection methods, the first is a gel electrophoresis based-method, while the second is bead-based size selection method For the gel-based method, the adapted library fragments are run on a gel to separate the fragments by size and the band corresponding to the size of interest is collected Using the bead-based method, magnetic beads are used with varying concentrations of buffers to isolate the DNA fragment sizes of interest Final library fragment size is important for efficient, high quality DNA sequencing downstream Bonus, when preparing amplicon libraries, size selection is usually not necessary, as long as the PCR products were already designed to be within the desired size range We are almost done! The last and very important step is library quantification and QC Accurate library quantification is important for successful template preparation and sequencing There are a few library quantification methods commonly used The first is analysis by the BioanalyzerTM system This method gives you both library concentration and fragment size information The second is qPCR This method provides the most accurate library quantification information, as it only measures amplifiable library fragments, but lacks library size information Which method you prefer is entirely up to you (and probably what’s available in your lab) And that’s NGS library preparation in a nutshell Of course there are variations on this theme depending on your application, for example, gene expression or DNA methylation analysis, but the fundamentals stay the same Since your library quality dictates the success of all downstream processes from template preparation to sequencing, understanding the library preparation is important to help ensure you get the highest quality sequencing data I hope this video was helpful on NGS library, and I am sure you’ll have more questions Submit your question at thermofisher com ask and subscribe to our channel to see more videos like this And remember, when in doubt, just Seq I


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