1. Shotgun sequencing can be performed usingproducts of PCR but it leads to concernsof incomplete or wrong PCR products. With cloning, the presence of suchincomplete or incorrect PCR products is eliminated. When fragments are clonedusing a host such a bacterium, it ensures that correct sequences are used forfurther processing. Cloning has a lower error rate as compared to PCR and henceproduces DNA sequences with maximum accuracy.
Cloning produces multiple copiesof the same fragment making it easier to sequence one known similar clone. Evenif PCR without cloning is doable, it only increases the risk of amplificationand expression of the incorrect gene. Another reason why cloning is preferableis because when multiple copies of one similar fragment have to be sequenced,only one type of primer can be used.
(PCR requires different primers fordifferent fragments). Cloning helps in proper expression of the required gene,its amplification and prevention of mutations. 2. De novo assembly is used to assemblefragments into a whole genome without any reference. Its basically the mergingof reads(fragments) to form a long sequence similar to the original genome fromwhich the fragments were obtained. De novo sequencing uses two basic algorithmsnamely OLC (overlap-layout-consensus) and de Bruijn graph. Reads are generated from different sequencing methods and areassembled using the De novo assemblers(algorithms). Certain reads producedthrough sequencing methods such as Illumina, 454 Roche, SOLiD systems etc.
,even though being higher order of magnitude per run, produce shorter reads ascompared to the classic sangers method. This makes the assembly computationallycostly. If the short reads have base calling errors, it’ll have an higherimpact. Next, the problem of repeat sequences is difficult to rectify if therepetitive sequences are longer than the short reads. 3. Cloningis done to carry out amplification of DNA fragments during formation contiguousstrands of DNA. When amplification is to be done for Illumina and 454,techniques such as Bridge PCR and Emulsion PCR and used substituting cloning.When talking about parameters that will ensure the proper functioning of theseamplification methods (substitute for cloning), the concentration of DNA is ofutmost importance.
Illumina uses a flow cell that has various nucleotidesattached. DNA fragments of a lower concentration have to be used so that the don’tovercrowd the clusters and lead to a mixed signal. Similarly, with 454, thenumber of beads and DNA concentration should always be adjusted to avoidunnecessary discard of the beads. If more DNA fragments are added as comparedto the number of beads, it might again lead to a mixed signal due to attachmentof more than one DNA fragment on the bead.
DNA fragment number can be estimatedby calculating using the molecular weight and average weight of DNA or by usingtechniques such as gel electrophoresis. 4. Theprocedure of illumina starts by cleaving the DNA to shorter fragments. Adaptorswhich are short DNA sequences are then ligated to the ends of the DNAfragments. These DNA fragments are then denatured with the help of sodiumhydroxide. The DNA fragments are then loaded on the flow cell and thecomplementary sequence binds to the oligonucleotides present on the flow cell.
The oligonucleotides are added to facilitate the attachment of DNA to the flowcell. One attached, the DNA sequences are formed with the help of polymerase.Post this the original strands are washed off. Now the reverse strand bends andthe top adapter attaches to another oligonucleotide.
DNA sequencing occursagain. This process is called as Bridge amplification. The DNA strands are thenseparated using heat. Clusters of identical strands are created. A mixture offluorescently labelled terminators i.e.
labelled dNTPs (A, T, G, C) are addedto the flow cell and are attached to the DNA strands with the help of DNApolymerase. These dNTPs are recorded as they produce a different colour and thesignal is generated. Reverse terminator nucleotides prevent the addition ofmore than one base on the DNA fragment. Once the fluorophore is cleaved andremoved, another set of labelled nucleotides can be added. 5. Sequencingmethods such as Illumina and 454/ion torrent produces shorter read lengths. Shorterreads can be generated due to the short length of input DNA or DNA damage ormodifications.
Other reason that limits the read length in technologies likeIllumina are inefficacy of the PCR due to error products or homopolymers orsecondary structures. One of the most important factors that limits read lengthis the Phasing problem. For example, in Illumina or Ion torrent, clusters ofidentical strands are sequenced together and have the same base incorporatedproducing one single signal.
If one or two of these identical strandsincorporate two bases instead of one, a mixed signal is generated. As thefurther cycles are continued, the more the fragments get out ofsynchronisation. The signal created due to this goes out of phase and overshadowsthe main signal making it difficult to interpret. 6. 454 (Pyrosequencing)Pyrosequencingis a sequencing by synthesis method which is based on the detection of pyrophosphates which arereleased during the sequence synthesis. The method utilizes various enzymessuch as DNApolymerase, ATP sulfurylase, luciferase and apyrase, in a mixture of which primer hybridizedsingle stranded DNA is incubated.
A sequential addition ofnucleotides A, T, G and C is done on the immobile primer which leads to theincorporation of particular primer to the binding end causing the release of apyrophosphate(PPi). The pyrophosphate is then converted to ATP by ATPsulphurylase. This ATP is then utilized as a substrate for the conversion of luciferinto oxyluciferin mediated by luciferase. This reaction produces visible lightwhich is detected and used for further analysis. Apyrase degrades the unusednucleotides and ATP. Oncethis cycle is completed, another round of nucleotides can be added.
Ion TorrentIontorrent sequencing is a sequencing by synthesis kind of a method of DNAsynthesis which is depended upon the detection of an hydrogen ion which isreleased during the formation of the DNA strand. Ion torrent sequencersconsists of a microwells on a semiconductor chip which is connected to a sensorbeneath the chip. These microwells contain single stranded template DNA copiesand DNA polymerase. These wells are then loaded or flooded with A, T, G or CdNTPs. If one of these nucleotides incorporate with the binding end of thetemplate DNA (covalent bond)to form the complementary DNA, it leads to therelease of a pyrophosphate and a postitively charged hydrogen ion which is thendetected by the sensor in the semiconductor chip. If no nucleotide is bound, nosignal is generated.
Post the generation of signal, DNA assembly is carried outwith the help of software platforms. 7. – DNA polymerase is used to bring about thereaction and single molecules are utilized hence the signal doesn’t degrade.(DNA polymerase replaces reverse transcriptase)- Producesgreater number of reads in a shorter run time.- Largeamount of samples can be sequenced due to the shorter run time- Thereis no need of DNA amplification (no PCR)- Candetect DNA as well as RNA base modifications- Produceslonger reads which simplifies process of De novo assembly- Producesaccurate, correct sequences avoiding fragmented and erroneous assemblyof DNA- Lowererror rate