How massive parallel sequencing will help You to increase Your success rate?
Celina Cybulska, Ph.D.23 April 2015
Development of high-throughput DNA sequencing technologies that omit time consuming and labour intensive steps have opened new possibilities in life sciences. The beginning of the 21st century brought forth a closure of the thirty-year domination of sequencing by the Sanger’s method. Next-generation sequencing technologies enable rapid generation of data by sequencing massive amounts of DNA in parallel using methodologies that overcome the limitations of Sanger sequencing. Using the “depth of sequencing” tool, experts from INVICTA Genetic Laboratory, performe their PGD procedures with high accuracy and reliability.
Since 1977, when Fred Sanger introduced sequencing technique, DNA sequencing technology has undergone intensive development. Sanger method, based on de novo synthesis of a DNA strand using dideoxynucleotides terminators, was a dominant method for the following three decades. It underwent continuous improvements like using of recombinant DNA polymerases or fluorescent dyes instead of radioactive isotopes. In the 10 years after the publication of the Sanger method, first automated sequencing techniques were introduced.
Despite its wide availability Sanger sequencing has restricted applications. One of the limitation is throughput, that is, the number of DNA sequences that can be read in each sequencing reaction. The requirement for electrophoretic separation of DNA fragments for reading DNA sequence, increases time and limits the number of reactions that can be run in parallel. Despite efﬁcient automation, Sanger instruments allow simultaneous electrophoresis in only 96 or 384 independent capillaries, what provides a limited level of parallelization.
Development of the Next Generation Sequencing (NGS) also called “massive parallel sequencing” enables sequencing of millions of different DNA sequences in a single run.
NGS significantly enhances accuracy of analysis due to its “depth”. This term refers to the average number of times a base pair is sequenced in a given experiment. Possibility of sequencing many of amplicons in a single reaction, allows us to obtain a wide coverage of the targeted sequence. Saying that a given fragment was sequenced with 20X coverage, means that the 100% of targeted region is covered with an average read depth of 20.
In the classic Sanger sequencing, in single reaction only one fragment, called “amplicon” is allowed for sequencing and sequence read is a sum of signals from a population of molecules from one fragment only. NGS technique used in the INVICTA Genetic Laboratory, allows multiple, independent reading of each molecule and multiple amplicons are allowed for sequencing in one reaction. Therefore, this approach enables correction of sequencing errors making the NGS technique highly accurate and trustworthy. We can compare Sanger sequencing to “one man reading a given page” (no one is going to correct errors made by the sole reader) versus NGS “millions of men reading the same page (errors made by one man are corrected by plenty of others).
Introduction of next-generation sequencing technologies for diagnostic laboratories caused a change in the format of their work. The challenge is in the interpretation of the huge amounts of data that reach millions or even billions of base pairs in a single experiment. Increasing the scale of the study, while preserving its accuracy, allows experts from INVICTA Genetic Laboratory to provide comprehensive and accurate diagnosis during PGD procedures.