Next Generation Sequencing Of Tomato Spotted Wilt Virus By Illumina Platform1

(PCR), which isolates small fragments of DNA that have a high degree of variability from

This is done through X-ray crystallography and NMR spectroscopy technology and will continue to be an essential method of testing various biological hypotheses3. DNA sequencing and genomic mapping is a newly introduced technology that decodes the individual chromosomes, genes and nucleotide sequences through new technologies, sequencing data, and computerized maps. The quickly

Highlight the base, take a screen shot of the waveform, and paste in a screen shot of this region of the DNA sequence waveform highlighting the start base here:

This method, as well as the Maxam and Gilbert method, for sequencing DNA are transforming the world of science, medicine and the views of people around the world.

It is the result of the cycle sequence reaction and cleans up. When the computer is ran each base pair (A, G, T, C) is represented by a color. Another letter will appear on the eletropherogram called N this happen when the computer can’t decide which base pair it is. (source) Many scientist used this not only is it a great source to use but it helps up learn about all kinds of species and were they come from. According to Picou, Kheterpal, Gilman using eletropherogram to find out things about Alzheimer’s Disease (Picou, Kheterpal, Gilman 2012, pg.2). The National Center of Biotechnology Information (NCBI) uses eletropherogram to provide genetic information. The NCBI also provides a GeneBank a public available DNA sequence which contains over 65 billion and 200,0000 species. However using those DNA sequences and putting them into a Basic Local Alignment Search Tool (BLAST) a program that explores all the available sequence databases of DNA. Once is goes through that BLAST there is a laser that will read it and there are different lengths. The different lengths if there is enough fragments a peak will come up. Then many peaks will be seen which represents your base pairs. A good peak will have very distinct peaks but a bad peak will be overlapping each other and low wavy peaks or have many N’s. Once you Once the BLAST in ran you will receive an Expected value (E) and it should be very low. The E represents the background noise that exists for matches between sequences. A query length will come up which is the number of base pairs in your sequence. Then an accession number will appear and a scientific name will come up which you can find a common name by searching it on the

This assignment is aimed to enable me identify an unknown sequence that has been provided. I have thereby used the DNA database, journals on cell division and medical publications in undertaking this assignment. To identify the sequence, application of theories and practical modelling is necessary for this kind of work. The sequence to be analysed is essentially the nucleotides present in cells sample under examination. Since each of the four typical nucleotides are available, chain extension continues typically until, by chance, DNA polymerase embeds a dideoxy- nucleotide which is indicated as hued letters rather than the ordinary de-oxynucleotide demonstrated as vertical lines. In the event that the proportion of typical nucleotide to the dideoxy forms is sufficiently high, some DNA strands will succeed in including a few hundred nucleotides before insertion of the dideoxy form stops the methodology.

If the lifetime of the polymerase is long enough, both strands can be sequenced multiple times (called ‘‘passes”) in a single polymerase read. Moreover, the polymerase read could be split to multiple reads (called subreads) by recognizing and cutting out the adaptor sequences. The consensus sequence of multiple subreads in a single ZMW yields a circular consensus sequence (CCS) read with higher accuracy. If a target DNA is too long to be sequenced only one time in a polymerase read a CCS read cannot be generated, and only a single subread is output instead. Whereas, the CCS sequences are usually generated by transcript sequencing due to its relatively short length. According, Pacific Biosciences developed an independently protocol, Iso-Seq, for long-read transcriptome sequencing, including library construction, size selection, sequencing data collection, and data processing. Iso-Seq allows direct sequencing of transcripts up to 10 kb without use of a reference

Let the agarose powder sit in the buffer for a few minutes. The beaker is covered with plastic wrap an placed in the microwave. Microwave the solution slowly with boiling it. As soon as the solution starts to boil, take it out and carefully mix it with the hot gloves on and continue to heat the solution until it is completely clear. Once the solution is cooled, add 3µL of ethidium bromide stock to the solution and mix it by swirling it. The gel is poured into the prepared mould that is taped on the ends and eliminate any bubbles. Place the comb on the negatively (-) charged side. After the gel solidifies, remove the comb and tape and place the gel into the chamber. Gently pour TAE buffer over the gel as the gel should be completely covered by 2-3 mm of buffer. If air bubbles form, gently displace them with a disposable micropipette tip. Droplets of prepared loading buffer are placed on a piece of parafilm paper. The ladder will consists of only the buffer because it will work as a measuring device to compare the DNA samples. 10 µL from each of the DNA samples will be mixed with the loading buffer using the micropipette tip. The positive control will be consist of 10 µL of water and the droplet of loading buffer. The samples including the ladder and positive control are added to a separate well in the gel. Once all of the samples are loaded into their own

Sanger Sequencing was introduced in 1977 by Frederick Sanger and has been extensively used for at least 35 years (10,11). Even now, it is still frequently used for small scale sequencing work. No breakthrough of whole genome sequencing techniques was made until the launches of Roche 454 GS20 (12) and ABI SOLID (13) in the mid 2000. Soon after that, Ion Torrent (14), PacBio (15) and Illumina (16) platforms have been introduced and quickly occupied the market of genomic sequencing. Not until recently, the third generation sequencing, such as the nanopore (17), has been commercialized and initially tested. A timeline of the development of DNA sequencing platforms was described in Figure 1.

Aiming to reduce costs and improve quality and efficiency, the New Generation Sequencers appeared. With the surging of NGS technologies, today it is viable to sequence in a particular subject with a suspected genetic disease or with tendency to that compound disease, up to thousands of genes at the same time (Pareek & Smoczynski 2011). There are different existing methods, however all shared similar features about Massive Parallel sequencing reactions. All of them required a pre-determined library sample preparation through DNA break-up and amplification by custom adaptors. Each library fragment is amplified on a solid surface (cell flow) by covalent-attached DNA linkers (oligonucleotides) which hybridize the library adaptors. This amplification creates millions of DNA clusters, each originated from a single library fragment. Each fragment will act as a single sequencing reaction. After this, each machine provides a raw data at the end of the sequencing run. This run data is a collection of DNA sequences that were generated of each cluster.

To sequence DNA it needs to be amplified first and this can be done in two ways; The Polymerase Chain reaction (PCR) and Cloning. Cloning isolates the DNA fragments from restriction enzymes and then combines the fragments with the vector. Once united with the vector the DNA fragments are then able to replicate to provide an unlimited amount of DNA for the experiment. PCR allows DNA to be multiplied millions of times in a couple of hours, PCR is used because it’s highly specific, easily automated and can amplify very small samples of DNA. The technique of PCR has had a huge effect on genetic disease diagnosis. To sequence the DNA, Gel Electrophoresis is used as it separates DNA fragments according to their size by running an electric current through the gel (Boehm). Mapping and sequencing the genome of organisms has allowed tests to be conducted, for example; gene tests, this is where individual genes or short lengths of DNA or RNA are tested for and Chromosome tests; this is where whole chromosomes or long lengths of DNA are tested for (National Human Genome Research Institute, National Institutes of Health 2014c).

Illumina is a less expensive way to sequence. At 99.9% accuracy, Illumina is able to provide short read lengths to be analyzed using Phred. Phred is a base calling algorithm that calculates the location of a fluorescent peak and records the base for that particular fluorescent label. This technology is not ideal due to portability and sequencing prep. The DNA still has to be amplified to create a cDNA library using bridge amplification. Illumina requires a polymerase and fluorescently labeled dideoxynucleotide triphosphates (ddNTPs) followed by Sequencing by synthesis (SBS). This is the final step in generating the wavelength reading of nucleotide bases. (White, et al. 2016)

The nucleotide sequence was provided by Prof Peter Smooker to get it analysed in all the ways that it is possible. It was submitted into NEBCutter tool [6] ¬ to obtain its protein sequence and is shown in Appendix 1.

To get high-quality clean reads, the assessment of sequencing quality and read processing were performed by fastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). The low-quality reads were removed using the same filtering criteria as described previously []. The clean reads were aligned on the F. vesca reference genome [1] using HISAT2 using default parameters [2]. Stringtie with higher accuracy and precision was used to generate transcript assembly [2]. Meanwhile, all unmapped reads derived from the above libraries were de novo assembled using the Trinity under default parameters to obtain comprehensive

Also, DNA extraction, PCR and Illumina MiSeq sequencing were done on the soil, was noted that they knew to do this process since it was done on a by a previous study (Manli Wu, 2017). Multiple copies of the extracted 16S rRNA were done using 2 different primers and then sequenced on the Illumina MiSeq (Manli Wu, 2017).