Genotyping, Index Sequence Capture (ISC) and Single-cell sequencing

Scientific Operations

The Genotyping, Index Sequence Capture (ISC) & Single-cell Sequencing team within DNA Pipelines Operations is a high throughput Illumina® library creation team specialising in producing libraries quickly and at scale from extracted material and single cells.

Index Sequence capture (ISC)

Using various automated liquid handling platforms and next generation library preparation kits, we first construct Illumina® whole genome shotgun (WGS) libraries from extracted DNA. We then hybridise these libraries to oligonucleotides that target the regions of interest within the genome, the exome. This targeted sequencing enables us to enrich specific genes, exons and/or other regions of interest to allow the sequencing reads to be dedicated to only the regions that are of interest to our customer. As the final library predominantly represents only the regions of interest, these can be sequenced to a much higher depth or at a decreased cost when compared to sequencing the entire genome.

Single cell RNA (scRNA)

Using various automated liquid handling platforms and next generation library preparation kits we construct Illumina® libraries from single cells. Using flow cytometry, the customer sorts cells into a 96 or 384 well plates and provides these to the team. The cells are then lysed and the RNA fraction converted to cDNA and amplified. The cDNA then undergoes a standard whole genome shotgun (WGS) library prep with one major difference; to preserve material the DNA is sheared using a cocktail of restriction enzymes rather than the standard sonification method. Single cell sequencing allows differences in gene expression between individual cells to be identified. Potential uses of this data include identifying rare populations and understanding of drug resistance.

Genotyping by Sequencing (GbS)

Using various automated liquid handling platforms and next generation library preparation kits we construct Illumina® libraries from extracted DNA. This pipeline uses a two-step PCR approach to first target the using specially designed primers. We have a range of designed targeting primer panels in house for different input materials. Multiplex Indexes are then incorporated to allow a higher number of samples to be sequenced simultaneously. Material designated for this pipeline can also be extracted from dried blood spots and/or go through a selective Whole Genome Amplification (sWGA) first within the Sample Management team in DNA Pipelines Operations. The GbS service is predominantly used by our Malaria research programme but can also be used as a QC for WGS data.