User prepared libraries

Library/pool molarity and amounts

  • Measure the concentration by Qubit or similar fluorometric method. Nanodrop is not adequate for concentration measurements.
  • The molarity of a library/pool [nM] can be calculated using the concentration values [ng/µl] and the library/pool fragment size [bp] with the following formula:

  • The minimal required amounts are in the table at the top. However, provide more if you can since it is not uncommon to require more if the performance of your library is suboptimal.
  • If your sample concentrations fall just below the given minima, please get in touch as we may still be able to run your samples if you do not require maximum sequence yields.

Quality control

  • We will test the quality and performance of your libraries by qPCR prior to clustering.
  • If we determine by qPCR that your libraries have relatively poor performance and we cannot achieve recommended clustering concentrations, yet we believe we can still obtain data, we will contact you before a run commences. In this instance, the run will only be performed at your own risk, and our data output guarantees will not apply.

Minimal requirements for pools to be sequenced on the respective machines are given in the table below. If you have higher pool molarity you can downscale the volumes accordingly (but not less than 15 µl).

Sequencer (workflow) Pool molarity (nM) Min. volume (μl)

MiSeq

 

5

 

   20

NextSeq

   20

HiSeq 3/4000/X

   20 (per lane)

Novaseq SP/S1 (Standard)

 5

70
Novaseq SP/S1 (XP) 20
Novaseq S2 (Standard) 90
Novaseq S2 (XP) 20

Novaseq S4 (Standard)

180

(50 for 1/4 flow cell)

Novaseq S4 (XP) 20

Multiplexing

If preparing your own libraries, please pay attention to index compatibility when preparing your libraries as follows:

  • all samples to be run in a single lane MUST have different indexes.
  • Compatible indexes must be chosen. e.g. - for 2 samples per lane, Illumina indexes 6 & 12 or 5 & 19. The following rule applies to choosing appropriate index combinations: Each index sequencing cycle, irrespective of index read length, must contain at least one base from each of the following two pairs: (G/T) and (A/C). For more information on choosing compatible index combinations, please see www.illumina.com.

Submission:

  • Submit only in 10 mM Tris buffer
  • Provide Bioanalyzer (or equivalent) profiles of your completed libraries. In particular, it is essential that you demonstrate that adapter dimers are not present in your library preparations. Note that for long-insert libraries (>500 bp), and samples to be run on Hiseq 4000, HiSeq X or Novaseq, adapter dimers must be COMPLETELY eliminated. Examples are provided below:

Acceptable library, showing minor amounts of adapter dimer ca. 120 bp.

Library with high levels of adapter dimer. Cannot be sequenced unless adapters are removed.

  • If you are submitting less than 16 libraries, enter their names, molarities, volume submitted and indexes  in the Sample submission form, and if more than 16- use the    Sample information table.
  • If you are submitting pools, indicate the name of the pool (which should be labelled on the Eppendorf tube it is submitted in), its molarity and volume submitted in the Sample submission form and list the individual libraries with their names and indexes in the Sample information table.
  • Index information should be provided in the following format:

 

 

 

 

 

     
         
         
         
         
         
 

 

Published Mar. 20, 2020 10:58 AM - Last modified Apr. 8, 2020 4:52 PM