Sample preparation

Sample Preparation Protocols and Requirements

LIBRARY PREPARATION TYPEINPUT MATERIALMIN AMOUNTVOLUME
TruSeq Nano DNA
(standard DNA sample prep)
gDNA 200 ngUp to 50 μl
Nextera XT
(small genomes and amplicons*)
gDNA of small genomes/ amplicons5 ng Up to 10 μl
Exome SequencinggDNA1 μg**Up to 50 μl
ChIP-SeqChIP DNA20 ngUp to 50 μl
TruSeq RNA
(standard RNA sample prep)
Total RNA/mRNA1 μgUp to 50 μl
ScriptSeq complete
(Stranded RNA sample prep with ribosomal depletion)
Total RNA1 μgUp to 50 μl
CEL-SeqaRNAPlease contact usPlease contact us
SMARTer and SMART-Seq sample prepsTotal RNA/mRNAPlease contact usPlease contact us

*For amplicon sequencing please contact us. Amplicons will be accepted only following DNA purification.
**For low input protocol, please contact us.

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.