High-Throughput DNA Sequencing
Scientific Operations
The High Throughput DNA sequencing team within DNA Pipelines Operations is a highly automated high throughput team specialising in producing libraries quickly and at scale from customer-supplied or extracted DNA. We normalise and cherrypick customer DNA samples and carry out library construction and pooling if required. Once library prep is complete the samples are sent to be sequenced on the Wellcome Sanger Institute’s fleet of Illumina® instruments including the NovaSeq 6000, HiSeqX and MiSeq.
- The library whole genome shotgun (WGS) preparation services provided by our group include:
- PCR WGS library preparation
- PCR free WGS library preparation
Processes
DNA Whole Genome Sequencing (WGS) PCR-based
Customer samples are normalised so that 200ng of input DNA enters the process. The DNA is fragmented to 450bp fragments using a Covaris LE220 prior to library construction. During library construction the fragmented DNA is purified ready for End-repair, A-tailing and Adapter ligation. The ligated libraries are then uniquely dual indexed (UDI) and amplified using PCR. These UDI amplified libraries are quantified using a fluoresence based assay and then pooled ready for sequencing on the Illumina® HiSeqX platform. This is operated as a high throughput library preparation pipeline processing thousands of samples each week, providing a reliable, consistent service to our customers and generating high quality libraries for sequencing.
DNA Whole Genome Sequencing (WGS) PCR Free
The WGS PCR Free process is carried out within the same team with a number of modifications to the protocol. Customer samples are normalised so that 500-1000ng of input DNA enters the process. The DNA is fragmented to 450bp using a Covaris LE220 prior to library construction. During library construction the fragmented DNA is purified ready for End-repair, A-tailing and unique dual indexed (UDI) adapter ligation with no amplification step. These UDI libraries are quantified using qPCR and then pooled ready for sequencing on the Illumina® HiSeqX or NovaSeq 6000 platforms.
