Crown Institute for Genomics

Sample submission for Next Generation Sequencing (NGS)

Sample submission procedure

  1. If you want to submit DNA, RNA, cells or lysate samples, or if you want to consult regarding the experiment design and analysis, please open a project request click here.

    If you already want to submit a sample that is ready for sequencing (“ready to run”) please continue to section 3.

  2. You will receive a calendar invitation to a consultation meeting (kick-off), to discuss your project with the relevant staff (always scheduled for Sunday mornings). To the PI: please be prepared to present your project including any preliminary data that would help determine the best project design. At the kick-off meeting we will collectively define an action plan, timelines and responsibilities.
     
  3. A purchase order must be available before sample submission. WIS users should place an order through the Internal Services website (service name is NGS). For instructions on how to create a purchase order click here.
     
  4. See our requirements below for amount and volume of DNA, RNA and Ready to Run samples.
     
  5. Download and fill in the relevant submission Excel sheet:
    • DNA submission excel sheet, click here.
    • RNA submission excel sheet, click here.
    • Ready to Run sample submission excel sheet, click here. For each Ready to Run sample, fill out a separate excel sheet.
    • Cell lysate sample submission excel sheet, click here.
       
  6. Prior to actual submission of the samples, send an email to: Samples. The e-mail must include:
  7.  
  8. Order ID number.
  9. The relevant excel.
  10. Bioanalyzer or Tapestation result as a pdf file (optional).
  11. Clearly write the name of the sample on the side of the 1.7 ml Eppendorf tube. Ensure that the name on the tube is IDENTICAL to the name in the submission Excel sheet (from section 5).
     
  12. To coordinate your sample submission with us please call 08-9345168.
     
  13. You will be notified once your sequence data is generated or if there is an issue that needs to be addressed.

 

Requirements for DNA and RNA library preparation

DNA specific requirements 

Sample preparation options

 

DNA Amount

Sample

volume (μl)

Sample

Concentration

(ng/μl)

INCPM DNA seq PCR Free 1.1-2.2 μg 55 μl 20-40 ng/μl
INCPM DNA seq 220ng - 1.1μg 55 μl 20-40 ng/μl
INCPM ChIP seq 2-10 ng up to 40 μl  
Agilent SureSelect XT 200 ng up to 50 μl  
Agilent SureSelect XT 3 μg up to 130 μl  
Illumina TruSeq DNA 1.1-2.2 μg 55 μl 20-40 ng/μl
Illumina TruSeq ChIP 5-10 ng up to 50 μl  

 

RNA specific requirements

Sample preparation options RNA Amount

Sample

volume (μl)

Sample

Concentration

(ng/μl)

INCPM mRNASeq 0.1 - 2 μg up to 25 μl  
Illumina TruSeq RNA 0.1 - 4 μg up to 50 μl  
Illumina TruSeq Stranded Total RNA 0.1 - 1 μg up to 10 μl  
Illumina TruSeq Small RNA 1 μg up to 5 μl  
Clontech SMARTer Ultra Low RNA 1 ng 1-9 μl  

 

DNA* and RNA submission criteria

Quality parameters DNA RNA
OD260/280 ≥ 1.8 ≥ 1.8
OD260/230 ≥ 2 ≥ 2
RIN number   ≥ 8**

* DNA samples produced by PCR reactions will be accepted only following DNA purification.
** Please note that Agilent RIN numbers are based on human, mouse, and rat RNA profiles. Other organisms’ profiles may not generate high RIN numbers, but may still be high quality. You should find the expected profile for your species and assess the quality.
 
Note that the user is responsible for the results in case the specifications above are missing or do not met the criteria.
 

Ready to Run samples (library/libraries prepared by the users) requirements

Please submit 20ul of 10nM library in a 1.7ml tube.

We will be happy to receive the Bioanalyzer or TapeStation results (pdf). For 10nM library concentration calculation please click here.

Ready to Run libraries have the chance of carrying a strong sequence or base specific bias as well as low complexity regions. Further, suboptimal adaptor ligation or enrichment steps might generate a large amount of constructs, which may not allow for successful clustering of the entire sequence on the flowcell.

Be aware, that the submitter is responsible for supplying a suitable Ready to Run sample. The Genomics platform cannot be held responsible for any decrease in yield due to sequence biases or low complexity regions as well as protocol intrinsic problems of self-made libraries.