Our services include initial project consultations and project design, nucleic acid extractions (from a range of substrates), library preparations, sequencing and bioinformatics analysis. Most of these services can be accessed on either in individual* or combinatorial basis.
*Individual access will be granted on a project by project basis
Sequencing
The SMCL NGS Hub currently operates Illumina MiSeq (x3), NextSeq & HiSeq 4000 sequencing platforms.
We can incorporate sequencing into library preparation workflows or as a sequencing only service.
For sequencing only services, we would request detailed information on the library preparation, QC and quantification results (this can be provided for an additional charge), platform to be used, and desired sequencing required (advice can be given on this).
NextSeq 500
Suitable for larger scale sequencing (e.g. large panel & transcriptomics), where speed and flexibility are required. Available in two output types, with read length flexibility
- Mid Output = 160M reads
- 150 cycle
- 300 cycle
- High Output = 400M reads
- 75 cycle
- 150 cycle
- 300 cycle
MiSeq
Suitable for targeted & small-genome sequencing, with quick run times. Available in two chemistry types, with multiple read lengths kits available
- v2 Chemistry
- 50 (S), 300 (S, M & N) & 500 (S & N) cycle
- Standard (S) = 15M reads
- Micro (M) = 4M reads
- Nano (N) = 1M reads
- Max read length 2x250bp
- v3 Chemistry
- 150 & 600 cycle
- Max read length 2x300bp
HiSeq 4000
Suitable for large scale projects that require high data output (e.g. whole transcriptome & whole exomes). Can generate upwards of 312M reads per lane, or 2.5 Billion per flowcell (across 8 lanes). Various read lengths available; 50, 150 & 300 cycle kits available (or variations of up to max of 2x150bp).
Sequencing on the HiSeq 4000 can be purchased as complete flow cells (i.e 8 lanes) or on a lane by lane basis (subject to current availability and throughput).
Nucleic acid extraction
Qiagen QiaSymphony SP Extraction
- Fully automated, ‘walk away’ system
- Cartridge driven, bead-based nucleic acid extraction
- Can process a range of sample types (Tissue, cell culture, FFPE, Blood, Buffy Coats)
- Up to 96 samples/run
Library Preparation
We offer library preparation services across a range of standard applications, including:
- Whole exome sequencing – NexteraFlex workflow
- RNAseq – both mRNAseq & whole transcriptome (including applications for processing low quantity/quality RNA samples)
- Targeted panels – capture or amplicon based (e.g. TruSight One clinical exome & Ampliseq panels)
- Methylation analysis – RRBS & TruSeq Methyl Capture EPIC Library
- Whole Genome preparations
The above list is by no means exhaustive, and we are happy to discuss and advise on bespoke sequencing projects. (N.B.: some special kit requests may be subject to a minimum project size.)
Sample/Library QC – Agilent Tapestation 4200
- Automated, ‘walk away’ sample processing for 1-96 samples
- DNA – genomic (200bp to >60Kb), D1000 (fragments up to 1000bp) & D5000 (fragments up to 5000bp) – available in both high sensitivity & broad range formats
- RNA – available in both high sensitivity and broad range formats, degradation measured through generation of RNA Integrity Number (RIN)
Bioinformatics
To ensure optimal efficiency and security of the data, all data flow from the sequencers to the users is automated through our on-site servers (2x Dell PowerEdge R430 with attached Dell PowerVault MD1400s for a total of 96 cores, 500 Gb RAM and ~100Tb of RAID10 storage). Standard fastq files are generated from the internal bcl Illumina files using Illumina’s own bcl2fastq package with all the recommended settings. Data is then returned via a password protected https link.
By default, we offer raw NGS data in the form of fastq files, however we can also offer bioinformatic analysis as an additional service.We currently have validated and best-practice bioinformatic pipelines in place for the following:-
- DNAseq variant calling: whether the NGS data is WGS, WES or custom panels (both amplicon and capture based) – alignment to the reference, variant calling and annotation/prioritisation of the results using GATK’s best practices.
- RNAseq Differential Expression analysis: we will provide alignment and read counts for all genes measured in the experiment, as well as the differential expression analysis between the experimental groups using a STAR/readCounts/DeSeq2/Limma pipeline (experimental group structure required prior to analysis).
- RRBS methylation bisulphite converted DNAseq: Bismark based pipeline for methylation levels in sequenced regions and differential methylation analyses (experimental group structure required prior to analysis).
- Custom analyses: any other analyses will be considered on a project by project basis.
Additionally, raw (and processed, if any) data will be stored for a period of 6 months by default. Data may be stored longer term, however additional costs may be incurred.
