Pacbio Sequencing Report

A lot of people are interested in figuring out their genetic heritage. With that information they seek to figure out if their is a root cause to their health problems or just to see where their family was from based off of their DNA. Sequencing and analyzing DNA for individuals was not always possible. But as new methods were created, DNA analysis services were sold to people so they could have a deeper understanding about themselves, like how ones body is effected by caffeine.

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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.

Two different DNA sequencing techniques were used in this study. Sanger sequencing is a form of DNA sequencing in which the target DNA is copied multiple times in fragments of varying lengths. The ends of the fragments are marked with fluorescent chain terminator nucleotides that indicate the end of the fragments. These fragments would then be aligned according to any overlapping segments that they shared. This allowed larger regions of DNA to be sequenced via capillary gel electrophoresis (Khan Academy, n.d.). This sequencing method was used to sequence the human genome, but has since then become one of the more expensive and less efficient way to sequence.

The initial step of Sanger sequencing, designing primer pairs for PCR, is often performed manually, with the aid of software such as Primer-Blast that can analyse only one genetic locus at a time, or PCR Suite, that can analyse more than one locus, but does not check for specificity. The manual design of primer pairs is especially cumbersome and prone to errors for long lists of genetic loci.

Trimmomatic v.0.36 –REF was used to trim reads by removing leading bases with Phred33 quality scores < 5, trailing bases with Phred33 quality scores < 3, using a sliding window of 4 bases and removing the 5’ terminal base if the average Phred33 score of the 4 bases was < 15, and completely discard trimmed reads with less than 36 remaining bases. The high quality reads were subsequently mapped to the pig genome Sscrofa build 11.1 using the Star software v.2.5.2a and default parameters while simultaneously adding unique read groups to the files – REF. Gene prediction coordinates (release 11.1.90) were obtained from Ensembl (http://www.ensembl.org) Samtools v.1.3.1 –REF was used to merge bams from the same sample and to convert bam files to sam files before HTSeq -REF was run with the reverse stranded option to calculate the number of reads mapped to each gene.

Figure 1.1: A schematic representation of Sanger sequencing. The Sanger method uses dideoxynucleotides that terminate newly synthesized DNA fragments at specific bases either A, T, C or G. Then, the resulting fragments are resolved by electrophoresis through a denaturing polyacrylamide gel in four parallel lanes, and the DNA sequence can be read (Rosenberg and Pascual, 2014).

High-throughput genome sequencing technologies are currently the important topic in the biology and medicine that allow us to look at thousands of sequence reads at a time. High-throughput and next generation sequencing technologies have largely been using for standard sequencing applications. Anyway these new technologies bring with many new challenges for biologist and bioinformatics people as how to process and interpret the massive amounts of biological data so as to achieve at biologically significant results. We presented a concise description of most commonly used sequencing technologies and the associated challenges below.

Shotgun Sequencing generally has two approaches which are Hierarchical Shotgun Sequencing and Whole Genome Shotgun Sequencing. But, method preferred by Human Genome Project is the Hierarchical Shotgun Sequencing method. In this approach, the genome is broken up into an overlapping collection of intermediate clones such as Bacterial Artificial Chromosome (BAC). Then, the sequence of each BAC is determined by Shotgun Sequencing and the sequence of the genome is obtained by mapping the sequences of BAC. Now, let’s focus on the processes that happen in this approach. First step is the source of DNA is mapped with some known genetic markers. Next, the genomic DNA is cut into smaller pieces called as sub-clones according to the genetic markers. This

Sanger sequencing is a technique to sequencing DNA and Applied Biosystems were the first to make it commercial. It has been widely used for more than 40 years and is still one of the best even today. "Next-Gen" methods have more recently replaced the original Sanger sequencing technique especially for sequences with more than 1000 data points. For smaller-scale projects, Sanger method is still most prevalent where sequences of 500 up to a 1000 nucleotides are common.

RNA sequencing (RNA-Seq) is revolutionizing transcriptome studies. It’s a highly reliable tool for measuring gene expression across the transcriptome. Also, it’s providing deep insight previously

Next-generation sequencing (NGS) has lately been adapted to diagnostic prerequisites and a growing number of diagnostic facilities offer an assortment of NGS-based services. However, variable NGS-based methodologies ranging from amplicon-based (targeted) NGS, whole exome (WGS) or whole genome NGS (WES) exist and are suited for different applications. As regards to tumor diagnostics in molecular pathology amplicon-based NGS predominantly is usually applied this is due to the facts that

Polymerase α is the first mammalian polymerase detected. It is a member of the B-family of replicative DNA polymerases. Its primary role is to further extend the RNA primer by addition of deoxy-nucleotides (dNTPs) into a total of 20-25 nucleotides of RNA-DNA primer. Generally, replicative DNA polymerases, polymerize dNTPs by catalyzing nucleophilic attack of the 3’-hydroxyl group of the primer onto the α-phosphate of an incoming dNTP aligned to the template strand. The accuracy of the polymerases is achieved by a confirmation of the correct base pairing of the incoming dNTP and the template strand according to Watson-Crick base-pairing. The accuracy of the primases is further enhanced (100 fold) by their exonuclease activity where in the event of a mismatch the growing strand is pushed back to the exonuclease active and the mismatched nucleotide is excised.

The 5’-terminus has a methylguanosine cap and the 3’-terminus consists of a poly(A) tail [7]. The genome contains five open reading frames (ORFs), see figure 1A.

The polymerase chain reaction or PCR for short can be used to create many copies of DNA. This allows the DNA to then be visualized using a dye like ethidium bromide after gel electrophoresis. The process has been refined over the years, however the basic steps are similar.