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Sequencing Libraries - FAQ

Sequencing Library starting material and quantity requirements.

 

Important Notes:

  1. For best library construction results please submit the recommended amount of starting material or more.
  2. The amounts specified work well for mouse or human derived nucleic acids, but other species may require different amounts. Please enquire if you are working with other species.
  3. In our experience the amount shown below is is the minimum amount of starting material which will result in adequate sequencing results. Please discuss with our technical staff if your starting material is limiting.
  4. Please note the submission requirements for plates:
    • Samples to be submitted in Axygen 96 FS-C plates, or equivalent. If these are not available in your lab, contact us as we may be able to supply one
    • Wells E12, F12, G12,  H12 to be left empty
    • Submit samples frozen
    • Samples must be normalized for concentration across the plate
  5. Submission of constructed libraries
    • Consult the Requirements Document for technical information about GSC-compatible libraries, including primer, adapter and index sequences. Not all commercially available kits are compatible with the GSC's pipeline.  Please confirm your library protocol prior to submission.  Examples of factors to consider:
      • The HiSeq X  instrument is only supported for 8 bp single indexing.
      • The NextSeq 500 has a minor restriction on index sequences that can be used when barcoding libraries.
    • The GSC cannot guarantee that constructed libraries will be accepted for sequencing.  Every effort is made to confirm compatibility prior to submission, however successful sequencing cannot be guaranteed. 

 

Starting material requirements:

Submission of Tissues for Extraction
Sample typePipelineSubmission requirements
FFPE Genomic

Scrolls: min 120 mm2 x 10µm, ≤ 120mm2 per tube/ up to 5 scrolls per tube

Cores: 2.5mm x (1-3mm)/ up to 2 cores per tube

FFPE Ribodepletion

Scrolls: min 120 mm2 x 10µm, ≤ 120mm2 per tube/ up to 5 scrolls per tube

Cores: 2.5mm x (1-3mm)/ up to 2 cores per tube
OCT Genomic or RNA Sections: 50µm x 10mm x 1mm, minimum of 4 sections
Fresh Frozen Genomic or RNA Sections: 50µm x 10mm x 1mm, minimum of 4 sections
Submission of Nucleic Acids for Library Construction
Sequencing
Library Type
Starting
Material
Recommended
Submission
Amount
Minimum
Amount
ConcentrationQuantification
Method
Quality
Assessment
Method
Quality
Value
Additional
Assessment

mRNA using strand specific protocols

(ssRNA-Seq)

Total RNA 750 ng

250 ng

(please enquire for non-mammalian samples)

>12.5 ng/uL

(volume 20-35uL)
Agilent Bioanalyzer
RNA nano chip
Agilent Bioanalyzer
RNA nano chip
RIN > 7 A260/280
A260/230
RNA-Seq "lite" Total RNA 100 ng 15 ng >5 ng/uL

(volume ≥3uL)
Agilent Bioanalyzer
RNA pico chip
Agilent Bioanalyzer
RNA pico chip
RIN > 7 A260/280
A260/230
Ribodepleted strand specific RNA-Seq Total RNA

750 ng

2 ug for FFPE RNA

250 ng

1.2 ug for FFPE RNA

>12.5 ng/uL

>34.3  mg/uL for FFPE

(volume 20-30 uL)

Agilent Bioanalyzer
RNA nano chip
Agilent Bioanalyzer
RNA nano chip
RIN > 7 A260/280
A260/230
miRNA Total RNA 750 ng 500 ng

>300 ng/ul

(volume >4 uL)

Agilent Bioanalyzer
RNA nano chip
Agilent Bioanalyzer
RNA nano chip
RIN > 7 A260/280
A260/230

PCR-Free Genome

gDNA 1.2 ug 750 ng

>30 ng/uL

(volume    25-40 uL)

Quant-iT dsDNA
HS Assay
Nanodrop intact in
agarose gel
A260/280
A260/230

FFPE Genomic

gDNA 500 ng 250 ng

> 10 ng/uL

(25- 40 uL)

Quant-iT dsDNA
HS Assay
Nanodrop A260/280
A260/230

Bisulphite

gDNA 2 ug 1.2 ug >30 ng/uL

(volume 25-40uL)
Quant-iT dsDNA
HS Assay
Nanodrop intact in
agarose gel
A260/280
A260/230
Exome and special capture (small-gap genomic) gDNA 2 ug 1.2 ug >30 ng/uL

(volume 25-40uL)
Quant-iT dsDNA
HS Assay
Nanodrop intact in
agarose gel
A260/280
A260/230
ChIP ChIP'ed DNA 5 - 10 ng

>150 pg/uL

(~5 ng in 35uL)

Quant-iT dsDNA
HS Assay
PAGE if possible NA
TCR/BCR Total RNA 750 ng 400 ng

>133 ng/uL

(3uL)

Constructed Libraries library depends on #
of sequence lanes
depends on #
of sequence lanes
> 10 nM Quant-iT dsDNA
HS Assay
Agilent DNA chip Expected size
range

Contact Information

For additional information or to arrange for a cost quotation, please contact us.

Diane Miller, Projects Team Leader
Genome Sciences Centre, BC Cancer Agency
Suite 100, 570 West 7th Ave
Vancouver, BC, V5Z 4S6
Email: dmiller@bcgsc.ca
Phone: (604) 707-5808
Fax: (604) 877-6085

Page last modified Nov 16, 2016