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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. 
  6. Submission Requirements for Chromatin Immunoprecipitation
    • when cells are submitted
      • Snap frozen pellets
      • Total of 1M cells (per sample), this is the recommended amount if all 6 marks are done for one sample
      • The 1M cells (per sample) can be submitted in one tube
    • GSC histone modification core marks
      • H3K4me1
      • H3K4me3
      • H3K9me3
      • H3K27me3
      • H3K36me3
      • H3K27ac

 

Starting material requirements:

Submission of Tissues or Cells for Extraction: All samples must be frozen at -80°C immediately after sectioning.
Sample typePipelineSubmission requirements
FFPE Genomic

Scrolls: min 120 mm2 x 10µm. For tissue surface area ≤ 120 mm2 up to 3 scrolls per 0.5 mL Matrix tube

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

Scrolls: min 120 mm2 x 10µm. For tissue surface area /≤ 120 mm2  up to 3 scrolls per 0.5 mL Matrix tube

Cores: 2.5mm x (1-3mm)/ up to 2 cores per Matrix tube
OCT Genomic or RNA Sections: 50µm x 10mm x 1mm, minimum of 4 sections supplied in 2 mL tubes
Fresh Frozen Genomic or RNA Sections: 50µm x 10mm x 1mm, minimum of 4 sections supplied in 2 mL tubes
Cells Genomic PCR Free Minimum #cells: 1x10e6
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
Low input RNA (Ribodepletion) Total RNA 50 ng 40 ng >4 ng/uL

(volume 10 uL)		 Agilent Bioanalyzer
RNA pico chip		 Agilent Bioanalyzer
RNA pico chip		 RIN > 7 A260/280
A260/230
Ribodepleted strand specific RNA-Seq Total RNA

200 ng

1.0 ug for FFPE RNA

125 ng

800 ng for FFPE RNA

>12.5 ng/uL

>80 ng/uL for FFPE

(volume 10 uL)

 Agilent Bioanalyzer
RNA nano chip		 Agilent Bioanalyzer
RNA nano chip		 RIN > 7 A260/280
A260/230
miRNA Total RNA 1 ug 700 ng

>70 ng/uL

(volume 10 uL)

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

PCR-Free Genome

 gDNA 1.0  ug

700 ng

>17.5 ng/uL

(volume   25-40 uL)

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

Chromium Genome

 gDNA 1 ug

 250ng

 > 25 ng/uL (10-40 uL

 Quant-iT dsDNA
HS Assay		 Nanodrop High Molecular weight (HMW) DNA avg > 50kb

A260/280

A260/230

FFPE Genomic

 gDNA 1 ug 500 ng

> 12.5 ng/uL

(25- 40 uL)

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

Genome shotgun low input (small gap)

 gDNA 120 ng 30 ng

>1ng/uL

(20- 30 uL)

Circulating Cell-free Genome

 gDNA 50 ng 5 ng 10 - 30uL

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 1 ug 500 ng >12.5 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.

Statement of Work Project Management Team
Genome Sciences Centre, BC Cancer 
Suite 100, 570 West 7th Ave
Vancouver, BC, V5Z 4S6
Email:
Phone: (604) 707-5900

Page last modified Nov 15, 2019