Both of these variants were flagged to have poor quality scores and would have been flagged by the laboratory for confirmation by Sanger dideoxy sequencing
Following preliminary population surveys via dideoxy sequencing
(Alter and Palumbi 2009), we designed two mtDNA markers for haplotyping.
However, requiring relatively low-throughput dideoxy sequencing
as a validation of high-throughput NGS interrogation severely limits the utility of NGS.
Sanger dideoxy sequencing
method was followed in which the primer: 518F-5'CCAGCAGCCGCGGTAAT ACG3' and 800R-5"TACCAGGGTATCTAATCC3' was used for amplification process.
Respiratory specimens from the 2 infected humans and the 5 dromedaries that were still positive at the second sampling were analyzed by dideoxy sequencing
as previously described (13); the nucleocapsid gene sequences of all dromedary samples were found to be identical.
Primer pairs were tested in a subset of individuals from each of the 16 species, and those that produced amplicons in all 16 species were further evaluated by dideoxy sequencing
Most traditional sequencing, or Sanger Dideoxy sequencing
, is performed on PCR products.
Sanger Sequencing, also called Sanger Dideoxy Sequencing
, is the controlled interruption of enzymatic DNA replication through the use of dideoxynucleotides in primer-directed DNA extension to produce discrete DNA fragments (11,41) (Fig.
The bi-directional dideoxy sequencing
of 20 receptor kinase domains and their adjacent regions revealed novel somatic mutations in fibroblast growth receptor 1 (FGFR1) and frameshift mutations in growth factor receptor alpha (PDFGRA).
was done using the sequenase protocol (USB, Cleveland, Ohio) with the following modifications: detergents Nonidet P40 and Tween 20 were added to the annealing, labeling, and termination mix to a final concentration of 0.
Next-generation sequencing technologies far exceed previous DNA sequencing technologies, such as Maxim and Gilbert or Sanger dideoxy sequencing
, both in the breadth of genomic real estate that can be sequenced in a single experiment and in the depth of coverage for each genomic region sequenced (resulting from multiple individual sequence reads generated), which allows for a superior limit of detection for low-level sequence variants.
The draft genome sequence was confirmed by selecting additional primers to generate [approximately equal to] 1 kb products across the entire sequence for direct dideoxy sequencing
(Genewiz, South Plainfield, NJ, USA), applying TaKaRa LA Taq polymerase with GC buffer (TaKaRa Bio, Otsu, Japan) to obtain products in areas that proved difficult to amplify because of potential secondary structures or elevated GC content.