Sample preparation

Generating high-quality data on the Illumina sequencing platform requires high-quality libraries. The TGC offers library preparation services for a variety of starting materials.

Library quality highly depends on the quality of the starting material. In order to provide the best service, before accepting the samples we require quality assessment of the starting materials, for more details please see our Sample quality requirements.

Library prep services:

Standard DNA preparation kits*:

  • TruSeq Nano by Illumina
  • NEBNext Ultra II DNA
  • Nextera XT (recommended for low input and amplicons)

* There are several fragmentations methods available: Covaris shearing (mechanical) and/or enzymatic. The appropriate fragmentation method will be selected by the TGC team according to sample quality, quantity etc.

High-throughput DNA applications include:

These protocols require special pre-sample treatment. Please contact us before sending your samples.

  • Amplicon / 16S
  • Chip-seq
  • Exome

Standard RNA preparation kits**:

  • TruSeq RNA Illumina (polyA based method, for high quality RNA only)
  • NEBNext Ultra II Directional/non directional RNA
  • SMARTer_Stranded RNA-seq (rRNA depletion based, suitable for low quality RNA )
  • SMARTer_Stranded Total RNA-seq Pico V2 (design for low input)

**There are several rRNA depletion kits available: RiboGone-Mammalian (Clontech), RiboMinus Transcriptome Isolation Kit Bacteria (Thermo Fisher) and NEBNext rRNA Depletion kit (Human/Mouse/rat). The appropriate depletion method will be selected by TGC team according to sample quality, quantity etc.

Long-read library prep services:

Currently this service is still experimental.

  • Oxford Nanopore DNA (amplicons/genomic DNA)

Single cell RNA-seq:

  • CEL-Seq2 protocol (Technion development) – up to several hundreds of cells.
  • The 10X Genomics Chromium – beginning from approx. 1000 cells.

 

Sample quality requirements:

Genomic DNA should be intact. If your DNA sample is degraded please contact us to coordinate suitable sample prep.
RNA integrity must be confirmed using the Agilent Bioanalyzer/ TapeStation/ similar instrument, or by running the sample on an agarose gel.
Sample purity:
OD260/280 = 1.8-2.2
OD260/230 ≥ 2.0

Please note that the user is responsible for the sample’s quality.

Library Preparation

Library preparation is the process by which an initial sample, typically genomic DNA or total RNA, is processed to become a library ready for sequencing.

In general, DNA is sheared randomly, creating blunt- end fragments. Blunt ends are then adenylated in preparation for adaptor ligation. Adaptors contain sample-specific indexes to individually tag each sample. Size-specific magnetic beads are used for fragment size selection. Enrichment of adaptor-bound inserts is then achieved by PCR amplification, thereby enabling sample quantification for loading onto the sequencer. Samples prepared from RNA are usually subjected to poly-A selection in order to select for mRNA specifically to be prepared for sequencing. RNA is fragmented, reverse transcribed to cDNA, and then undergoes a similar process to that of DNA sample preparations.

The adaptors that were ligated to fragments during sample preparation hybridize to the flowcell on which they are sequenced. These adaptors contain a unique 6-8bp sequence, known as an “index” or “barcode”, essentially “tagging” each individual sample and enabling for multiple samples to be sequenced together in a pool. Because index sequences are unique, individual samples are then able to be identified according to their assigned index during bioinformatic analyses.