Protocol for Multiplex Amplicon Sequencing Using Barcoded Primers

Abstract

Gene differences predisposing to atherosclerosis disease are important to identify and allow us to understand cellular processes involved in the progression and development of atherosclerotic disease. This protocol describes the experimental procedure for multiplex amplicon sequencing using barcoded primers. The procedures included in this chapter focus on amplicon library preparation (including polymerase chain reaction (PCR) conditions), DNA library quantitation options, next generation pyrosequencing with the 454 GS Junior system and data analysis. The process from DNA to sequencing data can be completed within 5 days.

Appendix

1.1 A.1 PCR Clean-Up Approach

There are numerous methods for purifying the newly amplified product following a PCR reaction. The aim is to remove any unused primers therefore reducing the chance of a chimeric product formation between two PCRs. This is of great importance when undertaking this approach to sequencing since the primer sets used from both of the aforementioned PCR reactions share certain sequence homology.

Column based PCR purification kits utilize an adapted silica membrane column to clean-up PCR products in three easy steps: bind, wash and elute. The products recovered are generally greater than 100 bp and depending on the kit used this can be up to 10 kb in size. This rapid approach requires very little laboratory equipment and is relatively cheap.

An alternative to the above post PCR clean-up process is the use of magnetic beads to purify the amplicon of interest. The overall process includes bead addition, separation, wash and elution. This approach is useful for high-throughput requirements. An addition benefit of this technique, in comparison to column based options, is the ability to size select the wanted product via altering the bead:DNA solution ratio. This is particularly useful when more than two products are present.

A third option is to run PCR products on a agarose gel before dissecting the wanted band and ultimately purifying the cut out with a gel purification commercial kit. This method also uses a silica membrane column to capture the amplicons prior to washing. This is mainly used as a last resort when there is unspecific binding present, such as when using bisulphite treated DNA, due to the lower recovery of DNA when compared to the above options.

1.2 A.2 Bidirectional v Unidirectional Sequencing

With the 454 GS Junior there possesses a capability to sequence an amplicon either bidirectionally or unidirectionally. As the name suggests, bidirectional sequencing includes reading the sequence in two directions (forward and reverse) and conversely unidirectional approaches produce reads in only one direction (either forward or reverse). Obviously when wanting to highlight possible variants in amplicons bidirectional sequencing provides a greater deal of confidence and accuracy when analyzing results. Alternatively, unidirectional approaches cannot read in both orientations however these will contain double the number of reads. It is therefore recommended to sequence bidirectionally when investigating rare single nucleotide polymorphisms (SNPs) and unidirectionally when analyzing a wide diversity of amplicons.

1.3 A.3 Amplicon Length Considerations

The average read length for the 454 GS Junior is 400 bp with longest reads being around 600 bp [8] including the two adaptor sequences. It should be noted that shorter than average amplicons (<300 bp) may result in excessive amplification during the emPCR stage when using the suggested conditions. This issue is avoidable by optimizing the emPCR conditions to contemplate for the desired shorter product.