Guideline for amplicon sequencing with Illumina (NGS library indexing of self-made Amplicons)
GENERAL GUIDELINES FOR SAMPLE PREPARATION
Amplicon sequencing with NGS is used to detect low-frequency variants, quantify mixed populations, and analyze multiple samples with small targeted genomic regions at scale. To perform amplicon sequencing we recommend to follow a dedicated amplicon primer design to enable simple indexing via a second PCR.
The DcGC offers:
- Sample indexing of self-made amplicons.
- Sequencing of self-prepared amplicon NGS libraries after indexing.
Depending on the complexity of the index design, pooling with other samples may be available for cost-efficiency, or physically separate sequencing units may be required. For assistance, please contact us.
CHECKLIST CRITICAL POINTS
Amplicon primer design for Illumina sequencing
PCR products can be easily prepared for sequencing by adding universal Illumina adapter sequences at the 5’ end of your target-specific PCR primers. We suggest to use the so called TruSeq unique dual indexing adapter design (UDI1) for this approach. When using a different indexing set-up we only support the sequencing of full lanes. Please reach out to us upfront to align your indexing scheme with our setup.
TruSeq amplicon primer design
P5_Tail: (Read 1)
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT [NNNNNN...] <sequence-specific-part> 3’
P7_Tail: (Read 2)
5' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT <sequence-specific-part> 3’
The P5 primer must include an NNN stretch (at least 6 bases) to ensure high-quality sequencing. For increased complexity, use a mixture of primers with varying NNN lengths.
Amplicon length consideration
- Initial amplicon length should not exceed the sum of read 1 and 2 minus 10 - 20 bp’s to achieve the full coverage with a minor overlap of both reads. For possible read lengths please see the section SEQUENCING below.
Ordering of indexing primers
For full adapter sets, we recommend ordering via IDT using our “Guideline to order indexing primers / adapters via IDT”. The DcGC also offers aliquoted ready-to-use indexing plates with the TruSeq design, available upon request. For ordering a few indexing PCR primers yourself or custom designs, refer to our UDI TruSeq 8bp indexing PCR primers (Set C) sheet: IDT_8bp_2018_SetC_TruSeqIndexingPrimerSheet.pdf . Ensure oligos with sample indexes undergo NGS-grade purification to prevent cross-contamination, and specify this in your order.
INPUT MATERIAL PREPARATION
Post-amplification sample preparation
- Remove PCR reagents and surplus primers.
- Required concentration: 2-5 ng/µl in a minimal volume of 10 µl (measured by Qubit or similar precision fluorometric methods).
- Equimolar mixing is required for multiple PCR products per sample (one NGS library made of multiple different PCR amplicons).
Input Material Requirements
- Amplicons with universal Illumina adapter tail or ready-to-load NGS libraries.
- Purified and eluted in nuclease-free water or EB buffer (10mM Tris, pH 8-9).
For good quality data amplicons or libraries 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.
Concentration and Volume
- Ideal concentration: 10-20 ng/µl; minimum: 1 ng/µl.
- Volume: ≥10 µl.
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.
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 Portal.
SEQUENCING
For amplicon sequencing the sum of read 1 and 2 of the chosen read length should exceed the initial amplicon length (without tails). A detailed overview of available sequencing options can be found here. Please specify the read length and sequencing system accordingly during online sample submisison.
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.
Please provide the sequence or DcGC-supported name of Index in the Notes field. 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
- Email us a file with the index set name (supplier, item number) and index set sequences so it can be added to our laboratory database (Rosalind)
- Afterwards we will provide you the naming format of the indexes
- Use the exact same names for the index combinations, separated by semicolon, when adding the index information to the “Notes” field of each individual sample. You can add additional information to the notes field, they should be separated by semicolon from the index name
- 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 Portal. Therefore, please send an email to genomecenter@tu-dresden.de with your contact details (name, working group, working group leader, your email address and telephone number). This will enable us to set up an account for you. After you have received an automatic email, 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 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 Portal. Documents with information about the preparation method and quality control of samples and libraries are provided for your project.
Footnotes
Unique dual indices (UDIs) minimize the impact of index hopping that can potentially occur during Illumina sequencing and results in the misassignment of reads to a different Illumina index. It uses unique identifiers on both ends of each Illumina NGS library.↩︎