Custom Amplicon Alignment
Protocol Overview / Introduction
In this protocol we discuss and outline the process of aligning custom amplicons using primers for high precision.
There are a few concepts you should understand in order to have a clear idea of the protocol’s functionality
An amplicon is a piece of DNA or RNA that is the source and/or product of natural or artificial amplification or replication events. It can be formed using various methods including polymerase chain reactions (PCR). In this context, "amplification" refers to the production of one or more copies of a genetic fragment or target sequence, specifically the amplicon. As the product of an amplification reaction, "Amplicon" is used interchangeably with common laboratory terms, such as PCR product.
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. It is required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA.
More importantly, specific primers are selected in order to produce (amplify) the targeted amplicons. Such primers are present in both amplicons and reads, and they’ll be used in order make a quicker and more accurate amplicon-read alignment.
Pairwise sequence alignment methods are used to find the best-matching piecewise (local) or global alignments of two query sequences. Pairwise alignments can only be used between two sequences at a time, but they are efficient to calculate and are often used for methods that do not require extreme precision (such as searching a database for sequences with high similarity to a query). 
is a string metric for measuring the difference between two sequences. Informally, the edit distance between two words is the minimum number of single-character edits (insertion, deletion, substitution) required to change one word into the other.
Reads must be paired-ended and the tested size has been of up to 150 x 150bp for the reads and 250bp for the targeted amplicon regions.
Some researchers are interested in rapid sequencing of certain regions of a genome. For example, exons of cancer-related genes can be screened for somatic mutation arisen in the tumour. Amplicon sequencing is one of the commonly used approaches for such task. In this approach regions of interest are amplified by PCR, and the resulted DNA fragments are sequenced on a nextGen sequencing machine. Each amplicon is defined by two primers, so the size and position in the genome are known for each amplicon. The protocol is designed for analysis of pair-end sequencing, when both ends of each fragment are sequenced. It is also assumed that the reads are stripped from barcode adapters.
In nutshell the protocol takes the sequencing data, aligns reads to the amplicons using primers for high accuracy and then clips such primers for further analysis of the aligned reads.
this tool was design for Illumina targeted amplicon sequencing where primers are left attached to the reads after demultiplexing.
Read our flowchart of the Protocol.
The Custom Amplicon Alignment Tool follows the following steps:
Section 1: Custom Amplicon Alignment
The purpose of this section is to show how to use the Custom Amplicon Alignment tool, understand the input data and the best practices to obtain optimal results.
The tool is in the Galaxy Toolshed, search for “GVL” or “Align Custom Amplicon”.
You’ll need 4 inputs:
#Name_of_amplicon length_left length_right amplicon_name
The output is a BAM file containing the alignment of the reads with the reference.
Although the Custom Amplicon Alignment Tool performs quality control checks and sequence trimming, it is possible to apply stricter filters and controls if considered necessary.
Quality control/Trimming tools:
 Amplicon - Wikipedia: http://en.wikipedia.org/wiki/Amplicon
 Primer - Wikipedia: http://en.wikipedia.org/wiki/Primer_%28molecular_biology%29
 Sequence Alignment - Wikipedia: en.wikipedia.org/wiki/Sequence_alignment
 Levenshtein Distance - Wikipedia: http://en.wikipedia.org/wiki/Levenshtein_distance
 Needleman-Wunsch Algorithm - Wikipedia: http://en.wikipedia.org/wiki/Needleman%E2%80%93Wunsch_algorithm