The principal aim of the Federation of African Immunological Societies (FAIS) Legacy Project (FLP) is to increase the human capacity for research in immunology and related sciences on the African continent by training 1000 new PhD students over ten years. See more – faislegacyproject.com
Selassie Kumordjie is an FLP awardee from Ghana from the Kwame Nkrumah University of Science and Technology. Selassie’s PhD project is entitled “Plasmodium falciparum genetic variations underlying acquisition of malaria infections among Ghanaian children”. Selassie chose Brown University in the USA under Jeff Bailey for her PhD exchange project.
Selassie Kumordjie Report
Training Report on Next Generation Sequencing (NGS) at Brown University- Rhode Island from
23rd August 2019-19th November 2019
Next generation sequencing has garnered attention in recent years. Conceptually the major difference between NGS and traditional sequencing methods eg. Sanger sequencing is its ability for parallel sequencing of more nucleotides in a single run, a concept referred to as high throughput.
NGS has an advantage of multiplexing many reactions, even different species in one run. It also does deep sequencing, allowing the sequencing of highly specific data
Although large scale genome analysis e.g. whole genome sequencing is essential for discovery projects, sequencing a subset of genes or genic regions using targeted sequencing panels is advantageous for routine molecular diagnostics.
The primary aim of my stay in Brown University was to using Next generation sequencing technology to perform targeted sequencing of Plasmodium falciparum drug resistance genes screening for specific Single Nucleotide Polymorphisms (SNPs) across the entire genome of the parasite. The molecular Inversion Probe (MIP) Technique was employed to prepare libraries before sequencing was done. This is a unique technique that leverages on using the multiplexing ability of NGS to put many target probes in a single run. The method was use to sequence Pf positive samples targeting 800 drug resistant genes and 1700 SNPs.
MIPs use the technology of Capture by circularization in a multistep protocol. a panel is first created by pooling all desired MIPs sets followed by 5’ end phosphorylation. Each panel can be used separately or combined to create a larger panel if desired. Combining sample DNA, MIP Panel, Polymerase and ligase, a single capture reaction is done per MIP panel per sample.
Reaction is incubated isothermally and MIPs bind to targets followed by extension by polymerase and formation of a single-stranded circle by ligase. Linear DNA and other unbound probes are removed by exonuclease treatment and captured products are amplified Universal primers and illumina sequencing adapters. Barcoded samples are then pooled into a single tube which is further cleaned by solid phase reversible immobilization (SPRI) beads and agarose gel purification
As part of the quality controls for library preparation, I was introduced to using new equipment like the fragment analyzer and digital droplet PCR for quantification.
The Nextseq 550 from Illumina was used for sequencing the samples and I was introduced to using custom primers made specifically for the MIPs method because of the nature of multiplex probes being targeted for sequencing.
NGS requires specific bioinformatics tools to extract information from raw sequenced data. Several pipelines have been developed in this regard and these work at 3 basic tiers. Sequence generation, alignment to a reference genome and interpretation of results. I used the MIPtools pipeline which was used to perform the sequence and generations and alignments and created VCF files for further analysis. The MIPanalyzer and vcfR packages were also used in R for analysis and interpretation of sequenced data.
Primer design was also a skill I acquired while in Brown. Recombinant Primer Amplification is a relatively new method employing target amplification isothermally. I used primer design skills acquired to design primer for this technique since the technique requires primes slightly different from the usual PCR primers.
I was also introduced to software like GIMP which is used to annotate diagrams and plots for better descriptions.