Guidelines for submitting user-prepared Illumina sequencing libraries to the DcGC

GENERAL GUIDELINES FOR SAMPLE PREPARATION

The DcGC offers Illumina sequencing of self-prepared NGS libraries. Depending on the complexity of the index design we can either offer pooling with other samples for cost-efficiency, or we are forced to offer physically separate sequencing units.

Furthermore it is important to verify that your index design is compatible with the used Illumina sequencing recipes. This should be clearly stated in the manufacturers protocols, and should be further discussed by reaching out to us before preparing and submitting NGS libraries.

CHECKLIST CRITICAL POINTS

Please check carefully the following aspects!

Sample indexing and construct design

For Illumina sequencing we support TruSeq and Nextera adapter chemistries. Currently, we are working with 8bp unique dual indices (UDI). When using a different indexing set-up we only support the sequencing of full lanes.

For full adapter sets we recommend to order them for simplicity via IDT following our Guideline to order via IDT. If you only need indexing PCR primers or aim for a custom design you can make use of these two small guides to make the oligo ordering process easier:

If you order oligos yourself, be aware of the following: Adapters or oligos containing sample indexes need to follow NGS-specific purification protocols to avoid cross contamination by the oligo manufacturers. Please make sure you state that in your order. If your adapter contains a T overhang, it is advantageous to include a phosphorothioate linkage between the 3’terminal T and the preceding C during synthesis (after annealing the top and bottom adapter oligonucleotides). This provides nuclease resistance for this base, diminishing the probability of adapter dimers.

INPUT MATERIAL

Type

The required input is a ready-to-load Illumina library (or library pool), purified and eluted in sterilized nuclease-free water. NGS libraries can be shipped individually or already combined as pools.

Amount

Ideal = 10 - 20 ng/ul of a dimer free NGS library with a fragment size > 200 bp’s up to maximum 700 -1000 bp’s. (Please be reminded the pure adapter sequence length of a classical Illumina library is around 120 bp’s.) For accurate quantification, we recommend using the Qubit (Thermofisher Scientific) or Fragment Analyzer (Agilent) system. We don’t accept Nanodrop measurements, as they are not accurate.

Volume

min.volume = 10µl

Please elute samples in nuclease-free water (NFW). Do not use DEPC water.

Sample quality control and quantification

For good quality data libraries (pools) should be free of index adapter dimers. These byproducts can severely affect sequencing output and thus need to be depleted before sequencing. Therefore, please provide an electrophoretic analysis of the submitted libraries. In case of contamination with adapter dimers - if necessary - we will perform bead-based depletion of dimers at your expense.

PREPARING ALIQUOTS FOR SEQUENCING

Submitted NGS libraries should have a minimum volume of 10 ul and a concentration > 10 ng/ul. If you obtain less material, please get in touch with us before submitting those samples.

Supported plastics for sample submission

Samples can only be submitted in 96-well plates, 384-well plates or 8-strip PCR tubes with single lids. Strips or plates that are fully compatible with our systems are Sarstedt strips (#72.991.002), Eppendorf 96-well twin.tec® skirted PCR plates (e.g. Cat.no. 0030128648) and 4titude 384-well FrameStar plates (Cat. no. 4ti-LB0384/C). These can be provided upon request. When submitting in plates please fill them column-wise w/o empty wells in between.

Please identify tubes/samples with clear and short labels, e.g. with the sample ID (created in Rosalind during online sample submission) or a running index clearly referenced under the corresponding samples submitted over the Rosalind Client Portal.

SEQUENCING

Standard sequencing of paired-end, 2x100bp or 2x150bp reads can be requested. The requested coverage is mostly application dependent and can be specified for each sample during online sample submission. Please see below the available sequencing systems and their minimal coverage per sample and capacities per FC compartment. Alternative read-lengths and indexing scenarios are possible upon request if full flowcells are requested.

Minimal coverage per sample

Minimal coverage / sample is 20 mio read pairs. In case of pools this can be reduced upon request. Specificities of the species, sample, or project design can influence the number of reads required. We are glad to provide guidance on sequencing needs.

FC types and their capacities

FC name Possible number of sequencing cycles 1 Number of lanes 2 Number of clusters / lane | FC [Mio] 3
SP 100, 200, 300, 500 2 375 | 750
S1 100, 200, 300 2 750 | 1.500
S2 100, 200, 300 2 1.850 | 3.700
S4 35, 200, 300 4 2.250 | 9.000
Table 1: Flowcell options on NovaSeq 6000

more information

Illumina NovaSeq Flowcell specs

minimal coverage per sample

We do only accept submissions for sequencing of full flowcells

FC types and their capacities

FC name Possible number of sequencing cycles 4 Number of clusters / FC [Mio] 5
MiSeq nano v2 300, 500 0.8 - 1
MiSeq micro v2 300 3 - 4
MiSeq v2 50, 300, 500 12 - 15
MiSeq v3 150, 600 22 - 25
Table 2: Flowcell options on MiSeq

more information

Illumina MiSeqFlowcell specs

BIOINFORMATICS ANALYSIS

We have established bioinformatic pipelines for diverse applications that generate both quantitative and qualitative information about the samples. As we are very limited in bioinformatics capacity we can only provide bioinformatic support on request for self-made libraries of TU Dresden employees. Please get in touch with us.

SAMPLE SUBMISSION

Please log in to Rosalind to enter all necessary project information making use of the “Project Description” field during the project set-up dialogue. Give all relevant details about project design, sample preparation method and how the samples are eluted and/or preserved. Sample-specific information such as concentration, volume, used preparation protocols etc. should be provided when the samples are added to the created project.

Please also indicate if you require a bioinformatics analysis during project setup.

When submitting ready-to-sequence NGS libraries we need the comprehensive details about the library prep protocol, the indexing scheme and NGS construct design (TruSeq, Nextera, custom etc.).

For submitted NGS libraries the library index combinations should be added to the “Notes” field for each individual sample. This is different between DcGC-supported and custom / commercial index sets.

If you have any questions or feel unsure about an information, please contact us by email.

In case you are using commercial index sets please provide comprehensive documentation by email. To avoid any misunderstanding we recommend to get in touch with us before starting to prepare or latest before submitting your samples. It will be essential to

  1. provide a file with the index set sequences so it can be added to our sample database
  2. uses the exact same names for the index combinations when adding the index information to the “Notes” field of each individual sample; and
  3. the sequencing mode and Flowcell requirements are clearly discussed.

If you have any questions or feel unsure about an information, please contact us by email.

To submit samples, you must be registered in our Rosalind client portal. Therefore, please send an e-mail to genomecenter@tu-dresden.de with your contact details (name, working group, working group leader, your e-mail address and telephone number). This will enable us to set up an account for you. After you have received an automatic e-mail, please log in to Rosalind with your login details. If needed, we can reset your login details.

Delivering or shipping of samples

Once the project and sample information has been entered into the Rosalind Client Portal, please put the samples in a small plastic bag labeled with your name and Rosalind project ID.

Samples can be personally delivered to the lab at the (CRTD, 3rd floor south), daily from 9-12h and 13-16h. If needed, contact us to deliver samples outside of this time window (+49 351 458 82362 or email us at genomecenter@tu-dresden.de).

Please deliver samples under suitable conditions! When shipping your samples, please use an adequate-sized box and ensure a proper amount of cooling material to hold the sample temperature until it reaches us. The package should be addressed to:

DRESDEN-concept Genome Center
c/o Center for Regenerative Therapies Dresden
Technische Universität Dresden
Fetscherstraße 105
01307 Dresden

For concentrations > 10 ng / ul ice or cool packs are sufficient. For longer transports, lower concentrated samples should remain frozen during the transport to avoid any degradation.

When submitting in plates please get in touch with us to select the right sealing material for tight sealing of your plates!

Following up project progress

You can accompany the status of your project in our Rosalind Client Portal. Documents with information about the preparation method and quality control of samples and libraries are provided for your project.

REFERENCES

Footnotes

  1. The cycles can be customized when running complete flowcells such as: single end sequencing with 100 bp’s or 2 x 50 bp’s etc.↩︎

  2. Lanes are compartments on the FC that are physically separated from each other.↩︎

  3. Clusters are distinct monoclonal colonies of amplified NGS libraries on the FC surface after the so-called cluster generation.↩︎

  4. The cycles can be customized when running complete flowcells such as: single end sequencing with 100 bp’s or 2 x 50 bp’s etc.↩︎

  5. Clusters are distinct monoclonal colonies of amplified NGS libraries on the FC surface after the so-called cluster generation.↩︎