Glossary

Amplicon Library - the output of the GS Junior or GS FLX+ Instrument. This output provides the input data to the GS Amplicon Variant Analyzer, which identifies both known and novel DNA variants.

AVA - Acronym for Amplicon Variant Analyzer application

Command Line Interface (CLI) - a means of running the software from the system command prompt.

Encoding types - (See Multiplexer)

Filters - When viewing Variant Frequency, filters allow you to focus the Variant Frequency Table display to specified minimum and maximum values.

MID - a unique identifier that is attached a DNA library to identify the library to which an individual read belongs. Allows multiple libraries tagged with different MIDs to be sequenced together, within an individual PTP Device..

Multiplexer - specifies the association between MIDs and Samples, i.e. how the MIDs should be used to assign reads to Samples. Depending on the design of the Amplicon libraries, Multiplexers allow four types of encoding: Primer 1 MID, Primer 2 MID, Both, Either.

Primer sequences - when defining Amplicons, you can define primers using a series of nucleotide characters (A, T, G, C, or N).

Project - the main container for an Amplicon Sequencing experiment. In it, you specify the Reference Sequence (s) to which the sequencing reads will be compared, in search for Variants - the Amplicon(s) that constitute the library(ies) you sequenced [and hence, the reads in the Read Data Set(s)] - the Variant(s) that you specifically want the software to search and report on - and the Sample(s) that constitute the organizational basis for the analysis.

Raw image - the data captured during a sequencing Run from the GS FLX+ or GS Junior Instrument fiber optic bundle camera. Consists of images of the PTP Device taken during each nucleotide flow, capturing the light released by the sequencing reaction in each well of the PTP Device.

Read Data Set - a group of sequencing reads derived from an Amplicon library. In a Project, Read Data Sets exist within a Read Group to help organize the data and are associated with pairings of Amplicons and Samples.

Read Length - the length of the sequence (number of bases) used for analysis.

Reference Sequence - the DNA sequence against which the sequencing reads are aligned by the alignment software. Reference sequences may only contain nucleotide characters (A, T, G, C or N) where N is ???. The software processes shorter Reference Sequences more quickly, however, users may create longer Reference Sequences by concatenating short sequences with “N” characters inserted between the Reference Sequence(s).

Sample - a virtual container that groups reads for analysis and reporting. A sample provides the inputs to the analysis and reporting software. You can define any number of Samples in a Project, each associated with one or more Read Data Sets and with one or more Amplicons.

Sample-Amplicon pair

Target - the part of the amplicon to be aligned to the Reference Sequence during processing. Primers are not part of the target, and must be trimmed before processing.

Ultra-deep sequencing: Sequencing the same target of DNA, using amplicons, many times to find rare mutations.

Variant - a variation in nucleotides relative to the Reference Sequence. The software identifies four kinds of variants: substitutions, deletions, insertions and required matches, and a defined variant that may include any of these types in any combination. You can define multiple Variants in a Project, each associated with a specific Reference Sequence. Any number of Variants may be associated with a given Reference Sequence.

Variant frequency - the frequency of Variants as reported by the xxx phase.

Variant Pattern - the constraints that define a Variant. The AVA software uses four types of constraints to define Variants: “Must match”, “Substitute base”, “Insert bases,” and “Delete bases.”