DNA library preparation

Sample quality control (QC)

All DNA and RNA samples delivered to sequencing laboratory for library preparation will be quality controlled. The aim is to ensure that sample is suitable for requested NGS experiment. For DNA samples the dsDNA mass of the sample is measured (Qubit or Picogreen assay). In some cases the DNA fragment size distribution is also measured (e.g. FFPE extracts). For RNA samples RNA/DNA ratio is measured to reveal DNA contamination (Qubit) and the RNA fragment size distribution is determined (Agilent).

Customer provides

  •  DNA/ RNA samples  with clearly marked unique sample ID
  •  sample information sheet including the sample ID and other required info.

Service includes

  • DNA quantitation using Qubit BR kit (Pico Green for larger sample batches)
  • RNA quantitation
    - Qubit RNA and Qubit DNA BR kit for RNA/DNA ratio
    - Bioanalyzer Nano or Bioanalyzer small RNA

1. Library preparation Classic

In library preparation the dsDNA of the sample is converted into Illumina compatible indexed paired-end library.  In the process DNA is randomly fragmented into 250-350bp fragments by focused sonication. Library preparation of fragmented DNA involves repair of 3' and 5' ends followed by the addition of a non-templated dA-tail before ligation to an. After ligation the library is PCRamplified using indexed primers.

Customer provides

  • 3000ng high molecular weight DNA, preferred 130 µl in 25 ng/µl concentration
  • a sample information sheet including the sample names and other required info

Service includes

  • Sample quality control
  • Shearing of the DNA with Covaris S-2
  • End repair, A-tailing and ligation adaptor
  • NEB Next DNA Library Prep Master Mix Set for Illumina
  • Quantitation of the library: Bioanalyzer HS / Caliper GX HiSens analysis
  • Index PCR:  12 cycle amplification, 48 indices available currently (single indexing)
  • Quantitation and fragment size distribution of the library
    - Bioanalyzer HS or  Caliper GX HiSens analysis

2. Library preparation by transposition (Nextera)

Nextera approach utilizes transposon activity in forming Illumina compatible paired-end libraries. In this assay DNA is simultaneously fragmented and tagged with sequencing adapters in a single reaction. The Nextera transposition is a near-random event on the template DNA molecules but may favour certain sequence regions over others. Libraries prepared with Nextera kits are compatible with all Illumina sequencers. The protocol requires only 20-50ng of DNA input. Note, that lowering the copy number of input DNA molecules decreases the diversity of the libraries thus affecting the sequencing results.

Customer provides

  • DNA samples clearly marked with unique IDs
  • Sample information sheet including the sample IDs and other required info

Service includes

  • Sample quality control
  • Tagmentation reaction from (10)-50ng DNA (Illumina NEXTERA protocols)
  • Index PCR: 5 cycle amplification, 96 indices available through dual index protocol
  • Quantitation and fragment size distribution of the library
    -  Bioanalyzer HS or Caliper GX HiSens analysis

3. Target enrichment: exome and custom regions

Roche Nimblegen EZ capture technology

In target enrichment regions of interest (exomes or custom regions) are selected for NGS sequencing using hybridization techniques. Our laboratory process is streamlined for Roche Nimblegen EZ capture technology . Nimblegen captures are based on 2.1 million short (60-90nt) biotinylated oligonucleotide probes complementary to the target region.  After hybridization the probe-target duplexes are enriched for using streptavidin coated magnetic beads. After subsequent post-capture amplification the resulting library is Illumina compatible paired-end library. Hybridization enrichment is not 100% selective but has assay specific fraction of off-target DNA fragments.

  • For exome sequencing the most appropriate kit is selected based on project needs.
  • For custom targeting an enrichment capture library is designed based on genomic coordinates delivered by customer.

Customer provides

  • Customer will fill in the sample information sheet and select the desired kit or provide the unique code of the probe design. Sample names must be unique.
  • Customer will apply for FIMM user account.

Service includes

  • Hybridization based enrichment of target regions
    - Roche Nimblegen EZ protocol with minor modifications
  •  Amplification and purification of the enriched library
    - Phusion master mix, QIAquick PCR purification columns and AMPure XP beads
    - Library quantitation with Agilent 2100 Bioanalyzer

4. Mitochondrial DNA libraries

Mitochondrial DNA enrichment has been tested for human, dog or mouse DNA samples using rolling circle amplification. Enriched library is further processed to Illumina sequencing compatible library using NEXTERA kits NOTE: only works on circular mtDNA!!

Customer provides

  • 10µl of genomic DNA in 10ng/µl concentration preferably on a 96-well plate.
  • Fill in the sample information sheet and select mitochondria sequencing box in the form.
  • Unique sample IDs
  • Apply for user account FIMM servers if nor existing already

Service includes

  • DNA concentration measurement with Qubit or PicoGreen assay
  • Mitochrial DNA amplificationt using Qiagen Repli-G rolling circle amplification assay
  • NEXTERA library preparation of the enriched mitochondria using Illumina Nextera DNA sample preparation kit
  • Library quantitation with Agilent 2100 Bioanalyzer
Last updated: 25.04.2014 - 15:37