Library Preparation
Library preparation is an essential part of any NGS experiment. There are many different library preparation methods suited for specific applications. Overall, we offer the preparation of libraries for these applications:
- Whole genome sequencing (incl. PCR-free)
- Targeted panel sequencing
- Amplicon sequencing
- Differential expression
- Full transcriptome sequencing
- Single-cell RNA-sequencing
Nanopore sequencing
CF Genomics offers selected methods as standard service. Others can be ordered and used on-demand.
For users preferring a hands-on experience with the library preparation (e.g. students who want to learn the method), supervised library preparation is possible . Kits used in standard library preparation methods are kept in stock and available for our clients.
DNA library Preparation
- xGen™ DNA Library Prep EZ UNI Kit: Suitable for many sample types with low-moderate amount of DNA, starting from 1 ng of DNA (including FFPE DNA)
- KAPA HyperPrep Library kit: with Covaris DNA fragmentation. Suitable for many sample types with low-moderate amount of DNA, starting from 1 ng of DNA (including ChIP DNA, buccal swabs etc.)
RNA library Preparation
- Lexogen QuantSeq 3’ FWD Library Preparation Kit. Library preparation designed for sequencing 3’ end of polyadenylated transcripts, suitable for gene expression experiments, Cost effective – low cost library preparation and requires lower sequencing depth. May be used also for degraded samples (eg. FFPE) and limited samples (<1ng total RNA).
- NEBNext Ultra II Stranded Library Preparation Kit with polyA selection/rRNA depletion. Stranded library preparation suitable for many different applications, including for non-polyadenylated transcripts with rRNA depletion. High quality intact RNA input required, low sample input (<10ng total RNA).
Targeted sequencing
Target enrichment
Prepared WGS libraries may be enriched for specific sequences (usually coding or regulatory regions) by using biotinylated probes. Different predesigned assays are available on the market (exome, disease related genes, pathway specific, mtDNA etc.) or custom assays with user defined content may be designed. We offer KAPA HyperExome library preparation as standard service; other target enrichments may be done upon request including various exome kits (Roche Nimblegen, Agilent, IDT) or PCR based assays.
Amplicon library
We offer preparation of amplicon libraries of one or more amplicons by two-round PCR method. The protocol is commonly used for the analysis of 16S, ITS, COX and other taxonomically important genes. Up to 384 samples can be multiplexed in one run. We do perform both rounds of PCR or only the second round after receiving PCR products containing nextera overhangs. The protocol follows the 16S sequencing protocol published by Illumina but can be easily modified for other targets as well and is usually sequenced on Miseq paired-end 300 cycles cartidge.
