Amplicon Deep Sequencing on the lllumina MiSeq/NextSeq Platform

Introduction

Amplicon deep-sequencing using next generation sequencing (NGS) technologies has become a powerful tool to study a wide variety of research questions. Typical applications include

  • CRISPR genome editing protocols of eukaryotes
  • Genome-wide transposon insertion analysis in microorganisms
  • Human leukocyte antigen (HLA) typing and screening of specific somatic mutations in tumor tissues

Common to all these approaches is that a PCR is used to amplify fragments which are then sequenced on a single-molecule level.

 

The Two-Step PCR Approach - How it Works

The two-step PCR approach combined with Illumina’s dual indexing strategy allows to process up to 384 samples in parallel (Figure 1). The first-step PCR uses primers containing a locus-specific sequence as well as a universal 5’ tail as specified in the Nextera library protocol from Illumina (Table 1). Instead of using only one single forward and reverse primer, some protocols make use of up to 3 forward primers that differ in length by adding wobble bases (Ns) between the locus-specific and common 5’ tail. This might be especially useful in high-throughput projects where the sequencing throughput is especially critical and many samples are pooled. However, for most projects the sequencing throughput is high enough by simply using one forward and one reverse primer. If you need further background about this particular topic please contact us. The resulting PCR amplicons are then used as templates within the second-step PCR for further amplification but also to include the indexes (barcodes) as well as the Illumina adaptors. The Illumina indexing strategy for the second-step PCR consists of 16 forward primers and 24 reverse primers (see also table 2 and 3). The combinatorial use of these primers (16 x 24) defines the maximal number of 384 samples which can be pooled and sequenced on one Illumina MiSeq/NextSeq run.

 

 

Figure 1: Overview of the double indexing strategy used in the Illumina two-step protocol. During the Illumina sequencing step the amplified genomic sequence including the specific primers (grey and blue bars) as well as the forward and reverse barcodes (orange bar) are read-out.

Table 1: 5’ tails used for the first-step PCR. Oligonucleotide sequences © 2007-2013 Illumina, Inc. All rights reserved.
Table 2: Indexed forward primers for the second-step PCR. Oligonucleotide sequences © 2007-2013 Illumina, Inc. All rights reserved.
Table 3: Indexed reverse primers for the second-step PCR. Oligonucleotide sequences © 2007-2013 Illumina, Inc. All rights reserved.

How to Order the NGS Primers?
Download first the specific “Order Form_IlluminaAmpliconDeepSeq” under “Related Downloads” (see grey column to the right). Specify now the locus-specific sequences for your first-step PCR primers and then select your desired indexed forward and reverse primers. Send the upload sheet to info@microsynth.ch and request your specific offer (volume discounts possible). Alternatively, directly order your oligos from our online webshop by observing the following rules:
  • Oligo Name: Use the prefix “NGS_” ahead of your oligo name
  • Scale: Select “0.2 μmol” as synthesis scale
  • Purification: Select “HPLC” as purification approach

Example of Primer Pipetting Scheme for 96 Samples (8 x 12 indexes)

Table 4: Pipetting scheme for barcoding 96 samples using 8 i5 indexes (vertical) and 12 i7 indexes (horizontal). Using the 16 x 24 described indexes of Illumina it is also possible to unequivocally indentify up to 384 samples.

PCR Design Considerations
In general, the first-step PCR is a standard PCR using a proofreading polymerase and 5’ tailed PCR primers. The only point to consider is the length of the amplified product including the locus-specific parts of the forward and reverse primers. If the sequence of the entire amplicon is of interest, the Illumina forward and reverse reads are stitched. This requires for overlap ≥50 bases of the forward and reverse reads (Table 5).

Table 5. Various Illumina read lengths speci¬fications and their corresponding maximal amplicon sequencing lengths. Single-end run configurations are also possible depending on your specific project. Please contact us to discuss these possibilities.

Microsynth Competences and Services
One of Microsynth’s core competences is in the field of amplicon deep sequencing. Microsynth is able to offer its customers a non-stop service covering the entire process from experimental design planning, DNA isolation, PCR amplification and sequencing up to bioinformatics analysis of the generated data for typical deep sequencing projects (Figure 2).

DNA Isolation: Either the customer provides isolated DNA or outsources this step to Microsynth (>13 years of experience in DNA/RNA isolation from various and demanding matrices).

PCR Amplification: The PCR amplification will use a state-of the-art high-fidelity polymerase resulting in high-quality multiplex amplicon libraries. The customer either provides the DNA, the first-step PCR products, or the second-step products. The advantage of providing the first-step PCR products is that you will only need two primers per locus and do not need to order any indexed primers. PCR products are purified, quantified with fluorescence spectroscopy using Picogreen and pooled in equimolar amounts.

NGS Sequencing: Sequencing is done using Illumina MiSeq/nextSeq sequenc¬ing technology. Both technologies allow high-throughput profiling at low costs and the MiSeq has the additional advan¬tage of long reads (up to 550 bp).

Bioinformatics Analyses: Depending on customer requirements Microsynth can offer customized analysis of the data including QC of the sequence data, stitching, demultiplexing, mapping and/or identification of indels and SNPs.


Figure 2: Typical steps in a amplicon deep-sequencing project. Depending on researcher needs Microsynth can deliver a non-stop service starting from DNA isolation, over PCR amplification and sequencing to customized data analysis. Further, customer either sends samples for DNA isolation, first-step PCR or second-step PCR products or pooled ready-to-sequence libraries.

Example Outputs for an Amplicon Deep-Sequencing Project


Figure 3: Example output of a deep-sequencing project. Sequencing reads of individual samples are aligned to the reference and the distribution of SNPs (3A) and Indels (3B) are reported. In addition, Veen diagrams are displayed to visualize the number of mutations shared by the different samples included in the analysis (3C).






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