Hi,
I heard that the ng sequencing is more accurate than the Sanger sequencing in generating sequences. For example I heard that if I want to sequence a bacterial gene that I previously amplified with the Sanger method, the final sequence might contain bases that are mutated because of the action of Taq polymerase. With the ng method this does not happen because you don't perform the PCR but sequence all the genome extracted by the bacterium.Even in case of mutations, the contigs will be generated based on the average of the sequence of the reads for that sequence. I did not understand very well what it means.. Do you have an idea?
Thanks in advance.
Every method has its advantages and disadvantages. Not all "next generation" methods are the same. 454, Illumina, PacBio and others are all "next generation" but each is different. The thing that "next generation" methods have in common is producing hundreds to hundreds of thousands of reads of the same region of DNA. This "depth of coverage" depends on what the input DNA was (hundreds of thousands of reads for a single short input DNA like a single gene being sequenced, hundreds of reads for each region in a complete eukaryotic genome).
PCR can introduce errors or mutations into a sequence, but it is not that common, and there are many ways to reduce or eliminate that problem. One way is to perform several PCR reactions (in parallel, not in series) so that if an error occurs in one it will not be seen in the others. This is essentially increasing the "depth of coverage".
All molecular methods can be prone to problems with contamination with DNA that did not come from the organism that was supposed to be sequenced. Many of the "reads" in any next generation sequencing project can be from contaminant DNA (a low percentage of total reads, but still many reads) so it is crucial to have a data cleaning process in place to detect them and eliminate them.
The error rate of reverse transcriptase, used for converting messenger (or viral) RNA to DNA has an error rate of roughly one base change per 10,000 bases. The polymerases used for PCR have different rated depending on whether or not they have been modified to remove proofreading activity. There are "high fidelity" polymerases and some with less fidelity but they all have an error rate that is better than the reverse transcriptase, something on the order of one mutation per 100,000 to 1,000,000 bases.
With PCR, the major worry is an error that occurs in the very first rounds of amplification if a single template molecule initiated the reaction. Any error in the first rounds or replication will then be copied in later rounds.
Dear Silvia Caprari,
Sanger sequencing is the gold standard sequencing technology, so results from other sequencing platforms are usually verified using Sanger sequencing. A Sanger sequencing run can be accomplished in about 5 hours at a cost as low as a few dollars per sample, so it is an accurate, fast, and low-cost method for small targeted sequencing studies.
While Next generation sequencing is used for whole genome sequencing or for Long reads which is very tough to get Sequence through Sanger Sequencing.
Sequencing of less than 100 target regions and confirming results from larger targeted sequencing studies is accomplished most effectively by Sanger sequencing.
More over i have processed number of bacterial and fungal identification samples using 27F & 1492R (for bacterial identification) and ITS (for fungal identification) universal Primers in my laboratory with the Instrument Abi 3730 DNA Analyzer and The Results were up to the Mark.
Regards,
Srinivas.
Sanger sequencing is still the most reliable approach for sequencing and precisely for single genes. You may sequence twice to be sure that your sequence is free of artefact mutations. NGS is used in case you aim to sequence the whole genome.
Here is a link resolving your query: When Do I Use Sanger Sequencing vs NGS?
https://www.thermofisher.com/blog/behindthebench/when-do-i-use-sanger-sequencing-vs-ngs-seq-it-out-7/
Good luck!
simply, I can say that Targeted resequencing by NGS machine takes short fragments of the DNA around 200bp to be sequenced, these fragments and each copy of it is sequenced then it has to be aligned with each other to generate the full fragment. think if you sequenced the same read more than 10 times could be a risk for nucleotide change only from Taq polymerase action?
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