Converting samples to sequence-ready libraries is often the rate-limiting step of NGS. The facility is able to perform the following library types for you - or help you to make your own libraries.
mRNA-Seq: The most common RNA-seq method in which poly adenylated transcripts are sequenced. The direction of transcription is preserved (stranded protocol). Good quality RNA is recommended (RIN score > 7).
Small RNA-seq: sequencing of smaller RNA (up to 35nt) including microRNA, piRNA and snoRNA species.
Total RNA-seq: whole transcriptome (including non-coding RNA) minus the ribosome. Tolerant of using poorer quality & FFPE derived RNA.
ChIP-seq: sequencing of ChIP DNA.
Whole Exome sequencing (WES) – sequencing of exome-captured DNA libraries.
Whole Genome sequencing (WGS) – sequencing of the total genome.
Methyl-seq: (bisulphite sequencing) for DNA methylation status.
Many specialised applications exist for NGS and the facility may be able to assist you with some of these libraries. See the following table for the input requirements for some common methods. Where a range is given, it is advisable that input amounts for all samples be matched to the highest amount possible. For RNA this will increase the chance of capturing low abundance transcripts and for both RNA and DNA help minimise PCR amplification duplication.
|Library Type||Stranded||Sample Type||Input (ng)||Volume (uL)||Quality|
|mRNA||Yes||Total RNA||50 – 1000||50||> 7|
|Small RNA||Yes||Total RNA||200 – 1000||10.5||> 7|
|mRNA low (Clontech Smart-seq v4 ultra low)||Yes||Total RNA||0.01 – 10||9.5||> 7|
|Total RNA||Yes||Total RNA||100 – 1000||10||> 4|
|16S||-||DNA||12||5||1.8 - 2.0|
|DNA Methylation||-||DNA||50 - 100||20||1.8 - 2.0|
|WGS or ChIP DNA||-||DNA||0.05 - 10||50||1.8 - 2.0|