High-Throughput RNA and Laser Capture Microdissection Biopsy (LCMB) Sequencing
Scientific Operations
The High Throughput RNA and LCMB Sequencing team within DNA Pipelines Operations is a high throughput Illumina® library creation team which specialises in producing libraries quickly and at scale from RNA and laser capture microdissection (LCMB)-derived material. Once library preparation is complete the samples are sent to be sequenced on the Wellcome Sanger Institute’s collection of Illumina® instruments including the NovaSeq 6000, HiSeq X, Hiseq 4000, Hiseq 2500 and MiSeq.
The High Throughput RNA and LCMB Sequencing team within DNA Pipelines Operations is a high throughput Illumina® library creation team which specialises in producing libraries quickly and at scale from RNA and laser capture microdissection (LCMB)-derived material. Once library preparation is complete the samples are sent to be sequenced on the Wellcome Sanger Institute’s collection of Illumina® instruments including the NovaSeq 6000, HiSeq X, Hiseq 4000, Hiseq 2500 and MiSeq.
The library preparation services provided by our group include:
- stranded mRNA libraries
- globin depleted RNA libraries
- rRNA depleted RNA libraries
- low input (10ng) RNA libraries
- Laser Capture Microdissection Biopsy (LCMB) libraries
RNA libraries
Using various automated liquid handling platforms and next generation library preparation kits, we construct directional Illumina® libraries from RNA input provided directly by our customers or extracted within the Sample Management team in DNA Pipelines Operations. Our aim is to provide our customers with an accurate and complete view of the transcriptome from low levels of RNA. Our standard library preparation uses oligo d(T) beads to pull down RNA containing polyA tails, isolating eukaryotic mRNA. During conversion to cDNA uracil is incorporated into one of the strands giving the libraries directionality. During the PCR step, unique dual indexes are applied to allow the samples to be multiplexed ready for sequencing.
We offer various modifications to the library preparation to suit the needs of our customers. These include an additional globin depletion or a ribosomal RNA depletion in place of the poly(A) pulldown. We also offer an optimised pipeline for very low levels of RNA, modified to include additional PCR cycles.
Alternative RNA preparation pipelines are available in other teams within DNA Pipelines Operations, with low quality or low input samples being processed within the Bespoke Sequencing team.
LCMB
Using various automated liquid handling platforms and next generation library preparation kits we construct Illumina® libraries from laser capture microdissection biopsy (LCMB) derived DNA, typically from 100-1000 human cells. Our aim is to provide our collaborators with high complexity genomic data despite the challenging nature of working with such small amounts of input DNA.