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Sequencing Libraries - FAQ

by Stephanie McInnis — last modified May 13, 2013 08:11 AM
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 AB1000 plates. If these are not available in your lab, contact us as we may be able to supply one
    • Wells E12, F12, G12 F12 to be left empty
    • Submit samples frozen
    • Samples must be normalized for concentration across the plate

 

Starting material requirements:

Sequencing
Library Type		Starting
Material		Recommended
Submission
Amount		Minimum
Amount		Concentration Quantification
Method		Quality
Assessment
Method		Quality
Value		Additional
Assessment
RNA-Seq Total RNA 10 ug

2.5 ug

for plants, 10 ug required

 >50 ng/uL

(volume 10-60uL)
 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
RiboMinus RNA-Seq Total RNA 5 ug 1.2 ug >25 ng/uL

(volume ≥10uL)		 Agilent Bioanalyzer
RNA nano chip		 Agilent Bioanalyzer
RNA nano chip		 RIN > 7 A260/280
A260/230
miRNA Total RNA 5 ug 1.2 ug >250 ng/uL

(volume ≥ 4uL)		 Agilent Bioanalyzer
RNA nano chip		 Agilent Bioanalyzer
RNA nano chip		 RIN > 7 A260/280
A260/230

Genome
(tube based or
small-gap plate-based)

 gDNA 2 ug 1.2 ug >40 ng/uL

(volume 10-40uL)		 Quant-iT dsDNA
HS Assay		 Nanodrop intact in
agarose gel		 A260/280
A260/230
PCR-Free Genome, large-gap plate-based gDNA 1.2 ug 0.7 ug

>12 ng/uL

(volume 30-60 uL)

 Quant-iT dsDNA
HS Assay		 Nanodrop intact in
agarose gel		 A260/280
A260/230
Genome, large-gap
plate-based
 gDNA 5 ug 2.5 ug >40 ng/uL

(volume 10-40uL)		 Quant-iT dsDNA
HS Assay		 Nanodrop intact in
agarose gel		 A260/280
A260/230
RNA-Seq from CDNA
 cDNA 100 ng 10 ng >300 pg/uL Quant-iT dsDNA
HS Assay		 Agilent DNA chip correct size A260/280
A260/230
Exome Capture gDNA 2 ug 1.2 ug >40 ng/uL

(volume 10-40uL)		 Quant-iT dsDNA
HS Assay		 Nanodrop intact in
agarose gel		 A260/280
A260/230
Special Capture gDNA 1.2 ug 800 ng >40 ng/uL

(volume 10-40uL)		 Quant-iT dsDNA
HS Assay		 Nanodrop intact in
agarose gel		 A260/280
A260/230
Exon Tiling PCR products 200 ng 100 ng >2 ng/uL Quant-iT dsDNA
HS Assay		 Nanodrop correct size/PAGE gel A260/280
A260/230
Amplicon PCR products 100 ng 50 ng >1 ng/uL Quant-iT dsDNA
HS Assay		 Nanodrop correct size/PAGE gel A260/280
A260/230
Whole Genome
Amplification		 gDNA 20 ng 10 ng >4 ng/uL Quant-iT dsDNA
HS Assay		 Nanodrop intact in
agarose gel		 A260/280
A260/230
MeDIP gDNA 2 ug 1.2 ug >40 ng/uL

(volume 10-40uL)		 Quant-iT dsDNA
HS Assay		 Nanodrop intact in
agarose gel		 A260/280
A260/230
ChIP ChIP'ed DNA 5 - 10 ng NA NA Quant-iT dsDNA
HS Assay		 PAGE if possible
 NA
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, Project Manager
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 May 13, 2013