Sample requirements

General Submission Advice

  • We will assess the quantity and quality of the DNA/RNA you submit upon receipt and inform you of the results. For this, we use the most sensitive/specific techniques available (Qubit for concentration measurements, TapeStation or Femto Pulse analysis for size distribution and checking for degradation). DNA and RNA samples for short and long read library preparation should be free of degradation.
  • Standard gel electrophoresis can be used to check the quality of your sample prior to submission, however, use of a Nanodrop to assess concentration can produce highly unreliable results so we would advise against this where possible.
  • If in doubt please contact us regarding your quality analyses.
  • For libraries prepared yourselves (‘Ready-prepared libraries for Illumina Sequencing’), see bottom of page.
  • Please contact us if you would like to discuss using any protocol other than those listed below. Similarly, if you are wondering which sequencing protocol would be most suitable for experimental goals please don’t hesitate to get in touch for advice.

 

Illumina Service Requirements

  • There are several Illumina library preparation services offered by the facility, with different requirements:

Method

Sample type

Concentration req.

Suggested volume

Amount req. per sample

Quality recommendation(s)

TruSeq DNA PCR Free

Genomic DNA

>30  ng/µl

60 µl

1.8   µg

DIN 5

TruSeq DNA Nano

Genomic DNA

>7.5 ng/µl

65 µl

488  ng

DIN 5

Illumina DNA prep (tagmentation)

Genomic DNA

>12  ng/µl

35 µl

420  ng

DIN 5

Illumina DNA PCR Free (tagmentation)

Genomic DNA

>50  ng/µl

30 µl

1.5   µg

DIN 5

Nextera DNA XT

Genomic DNA

>5.0 ng/µl

10 µl

50    ng

DIN 5

TruSeq Stranded mRNA

Total RNA

>30  ng/µl

55 µl

1.65 µg

RIN 7

Illumina Stranded mRNA - ligation

Total RNA

>20  ng/µl

30 µl

600  ng

RIN 7

Illumina Stranded Total RNA - ligation

Total RNA

>85  ng/µl

20 µl

1.7   µg

RIN 7

Illumina Stranded Bacterial RNAseq - ligation

Total RNA

>85  ng/µl

20 µl

1.7   µg

RIN 7

 

Oxford Nanopore (ONT) Service Requirements

  • There are four main Oxford Nanopore library preparation services offered by the facility, with different requirements.
  • The standard DNA ligation protocol can be performed with shearing and size-selection or without. Typically, shearing and size-selection produces more data but requires more input DNA.
  • We recommend you follow the guides provided by ONT when extracting samples for submission. Guides for several different species and tissue types are provided at the following link (you may have to register): https://community.nanoporetech.com/docs/prepare/extraction_protocols

Method

Sample Type

Concentration req.

Suggested volume

Amount req. per sample

Key QC requirement(s)

Ligation

Genomic DNA

≥40 ng/µl

~50  µl

≥2.0 µg

DNA Integrity Score (DIN) > 7.0

 

Ligation with Shearing and Size selection

Genomic DNA

≥25 ng/µl

 

~400 µl

≥10 µg

DNA Integrity Score (DIN) > 8.0

 

Rapid

Genomic DNA

≥50 ng/µl

 

~20  µl

 

≥1.0 µg

<30kb fragment size only

Ultra-long

Genomic DNA

≥40 ng/µl

 

~1.0 ml

≥40  µg

Most species require ≥50% over 50kb fragment size – can be discussed

Direct RNA

Total RNA

≥50 ng/µl

~20  µl

≥1.0 µg

RNA Integrity Score (RIN) > 7.0

 

PacBio Service Requirements

Method

Concentration req.

Suggested volume

Amount req. per sample

Key QC requirement(s)

HiFi

50 ng/µl

~250 µl

12500 ng

(12.5 µg)

DNA Integrity Score (DIN) > 8.0

Ultra-low

5.0 ng/µl

~15 µl

50 ng

DNA Integrity Score (DIN) > 6.0

IsoSeq (RNA)

50 ng/µl

~15 µl

750 ng

RNA Integrity Score (RIN) > 7.0

Amplicon

~20 ng/µl

~50 µl

1000 ng

(1 µg)

per pool or sample

Appropriate size distribution (PCR products should be similar sizes)

 

Ready-prepared Libraries for Illumina Sequencing

  • We can sequence Illumina libraries you have prepared yourself. Please pool and prepare an aliquot appropriate for the intended flowcell, as specified in the table below
  • Please let us know if the libraries require the addition of custom read or index primers to the flowcell for the sequencing run. Please submit your primers at 100 µM in 30ul.
  • At present, we only accept user prepared libraries for Illumina platforms and flowcells. This may change in the future. If you would like to submit a library you have prepared yourself for any other platform please contact us to discuss your requirements.

Flowcell

Concentration req.

Minimum volume

Key QC requirement(s)

MiSeq

5nM

50 µl

<1% Adapter dimers

NovaSeq SP/S1/S2

5nM

100 µl

<1% Adapter dimers

NovaSeq S4

5nM

200 µl

<1% Adapter dimers

NB. for multiple flowcells, please submit multiples of minimum volume.