For research use only
Publication Number MAN160424001 Rev. 0.1
April 24, 2016

This manual describes VariantPro™ amplicon design files. Up to 5 text files may be included in each design.

1 01_UserEntries.txt

This file provides a record of user specified design, sequencing, and target information.

2 02_TargetCompiled.txt

This file provides compiled target list. The list is produced by combining overlapping and closely located user specified target entries.

3 03_AmpliconList.txt

This file provides information on amplicon designs. Figure 1 below defines the locations of various sections of the amplicons. Each amplicon is flanked with two barcoded library primer sections. The library primers are compatible with the sequencers of user’s choice. The section between the library sections is probe. The probe is delineated by specific primer sections (specific primer 1 and specific primer 2), between prbStart and prbEnd, and is the captured section of corresponding target sequence. While the sequence reads of the specific primer sections are dictated by corresponding primer designs, sequence reads of between prbVarStart and prbVarEnd are dictated by sample DNA. While in Figure 1 a complete target as defined by tgtStart and tgtEnd is covered by a single amplicon, multiple amplicons arranged as tiles may be required to cover a long target (not shown in the figure).

VariantPro

Figure 1 Schematic illustration of amplicon sequences.

 

Probes can be in either ‘+’ or ‘-‘ strand, which is computationally decided by VariantPro™ amplicon design program. In pair-end sequencing, read 1 always starts from library primer 2 which carries an i7 barcode and read 2 always starts from library primer 1 which carries an i5 barcode. In 03_AmpliconList.txt, prbStart is always less than corresponding prbEnd. The molecular tags shown in Figure 1 may not be included in some panels.

4 04_Coverage.txt

This file provides amplicon as well as read coverages information. The document contains a full list of user specified target or variant regions with corresponding amplicons, amplicon coverages and read coverages.

5 05_OffTargets.txt

This file provides off-target information. Sometimes a target or a primer binding region is homologous with one or more regions in the same genome. Then, it becomes possible that the homologous regions are captured along with the intended target resulting in off-target amplicons. This data file identifies the genomic locations of these off-target regions, the size of the off-target amplicons, the estimated relative amount of the off-target amplicon as compared to on-target amplicon, and responsible amplicon-bound primers.

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