The processing and sequencing of amplicons is quite flexible and allows for a wide range of experimental design. A researcher can choose a variety of options regarding design parameters, such as the length of amplicons, the number of amplicons pooled together, the number of reads desired for a given amplicon pool, and whether to read from the A end, the B end, or both. Although the setup for a given experiment will depend on the specific project goals, there are a number of general guidelines that will ensure the best possible result.
- The highest confidence in low frequency variation will result from bi-directional reads.
- A high-fidelity polymerase must be used in the amplicon generation step. Use of a low fidelity polymerase will result in many amplification induced variations in the sequence. Although there are many choices of enzyme, Roche's FastStart High Fidelity PCR System has high fidelity coupled with robust amplification of a wide array of input templates.
- Greater confidence in results may be achieved by running replicates of the biological material through the sequencing process and comparing the results.
- The level of multiplexing should be determined by:
- Number of amplicons of interest
- Desired sensitivity or depth of coverage
- When sequencing mixtures of multiple amplicons, care must be taken in quantification and pooling of amplicons as equimolar mixtures will generate best results.
- Forward and reverse reads will eliminate most systematic, context-dependent sequencing errors.The ideal experiment has reads covering the amplicon forward and reverse.
The number of amplicons that can be combined in an experiment, while theoretically unlimited, is primarily determined by the desired sensitivity of detection. These following guidelines are a general, though conservative, aid to help determine the level of oversampling required for a desired level of detection. The following guidance accommodates experimental realities such as variation in quantitation, pooling, amplification or sequencing efficiencies of MID labeled amplicons, and amplification efficiencies of long versus short amplicons.
- Heterozygote detection – 40x coverage
- 5% variation of single base changes and multibase deletions – 1000x coverage (good statistical chance for 50 variation reads)
- 1% variation of single base changes and multibase deletions – 5000x coverage (good statistical chance for 50 variation reads)
- Single-base indels may require additional depth
Amplicon Sample Requirements
The quality and quantity of the DNA sample are critical to the success of this procedure. Any contamination in the starting material will be directly reflected in the output library.
Since it includes an amplification step, this procedure requires less input DNA than the other DNA library preparation procedures, though the amount required will depend on the nature of the experiment. For example, if searching for low abundance sequence variants out of a complex sample (such as genomic DNA), one should start with 5–20 ng of DNA. If the starting material is cloned into a plasmid or is a PCR–generated DNA fragment, 1–2 ng is usually sufficient. Ideally the DNA should be checked to ensure it is derived from the target organism and contains no other contaminating DNA.
At a minimum, the DNA sample should meet the following criteria:
- DNA should not be degraded, and should contain no particulate matter
- Input DNA size should be sufficient to allow amplification of the target
- OD260/280 should be approximately 1.8
- Concentration should be 5 ng/μl or above, in TE (0.5 ng/μl for cloned or PCR–generated targets)
Because DNA quantitation using OD260is variable and dependent upon DNA purity, the sample DNA concentration should be checked by fluorometry or gel electrophoresis on a 1–2% agarose gel using a DNA Mass Ladder.
The procedure for preparing a DNA sample for Amplicon Sequencing consists of a simple PCR amplification, but requires special Fusion Primers, which must be designed by the user according to the specific requirements of the experiment. Amplicon fusion primers must contain a directional GS FLX Titanium Primer A or Primer B sequence (which includes a four–base library “key” sequence) at the 5–prime portion of the oligonucleotide in addition to the template specific sequence at the 3–prime end. An optional Multiplex Identifier “MID” sequence may be added between the Primer A (or Primer B) and template specific sequences to allow for automated software identification of samples after pooling or multiplexing and sequencing (also referred to as “barcoding”)
The choice of appropriate PCR primers for the generation of the Amplicon library is critical for a successful experimental design, as studies aimed at identifying and quantitating sequence variants can only be as accurate and unbiased as the original amplification.
The 5'–part of each Fusion Primer, Primer A and B, is always the same, as dictated by the requirements of the Genome Sequencer System. The requirements are as follows:
- Length: 25 nt
- Primer A: 5' – CGT ATC GCC TCC CTC GCG CCA TCAG–MID – template specific sequence – 3'
- Primer B: 5' – CTA TGC GCC TTG CCA GCC CGC TCAG–MID – template specific sequence – 3'
- bind to the DNA Capture Beads of GS emPCR Kits
- anneal the Amplification Primers of GS emPCR Kits
- anneal the Sequencing Primers of GS em PCR Kits
- end with the sequencing key TCAG used by the system's software for base calling and to recognise legitimate library reads.
The 3'–part of each Fusion Primer is specific to each Amplicon:
- Length: typically 20–25 nt (may vary)
- Sequence: specific to each side of the desired Amplicon
- anneal to either side of the target to be sequenced
- serve as PCR amplification primer, during library preparation
- start the sequence of the reads, since they are fused directly to the sequencing key from Primer A or Primer B
- used by the Amplicon Variant Analyzer software to assign reads to corresponding Amplicons
- Design considerations:
- The normal constraints of PCR primer specificity and annealing conditions apply.
- It is recommended that the total length of the amplified products (including Fusion Primers) are between 200 and 600 bp.
- Total amplicon length should be less than 800 bp to facilitate high quality sequencing.
- When possible, design amplicons to cover the sequence of interest within the first 400 bp of sequencing; i.e., the first 400 bp after the adaptor sequence but including the key and both MID sequences (if applicable).