Custom/PCR Amplicon
The Custom/PCR Amplicon workflow evaluates short regions of amplified DNA (amplicons) for variants. The focused sequencing of amplicons enables high-coverage sequencing of particular regions across a large number of samples. The main output files generated by the Custom/PCR Amplicon workflow are .bam files (containing the aligned reads) and .vcf files (containing the variant calls). The Custom/PCR Amplicon workflow supports multiple manifests and consensus sequence reporting for multi-manifest runs.
The Secondary Analysis Summary pane provides a four graphs, described below.
Low Percentage refers to run statistics that should be near 0 for an ideal run.
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y-axis |
x-axis |
Description |
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Percent |
Mismatch 1 |
The average percentage of mismatches for Read 1 over all cycles. |
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Mismatch 2 |
The average percentage of mismatches for Read 2 over all cycles. |
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Phasing 1 |
The percentage of molecules that fall behind (fail to incorporate a base) in a sequencing cycle in Read 1. |
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Phasing 2 |
The percentage of molecules that fall behind (fail to incorporate a base) in a sequencing cycle in Read 2. |
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PrePhasing 1 |
The percentage of molecules that run ahead (incorporate an extra base) in a sequencing cycle in Read 1. |
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PrePhasing 2 |
The percentage of molecules that run ahead (incorporate an extra base) in a sequencing cycle in Read 2. |
High Percentage refers to run statistics that should all be close to 100% for a good run.
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y-axis |
x-axis |
Description |
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Percent |
|20/|1 1 |
The ratio of intensities at cycle 20 to the intensities at cycle 1 for Read 1. |
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|20/|1 2 |
The ratio of intensities at cycle 20 to the intensities at cycle 1 for Read 2. |
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Align 1 |
The percentage of clusters that aligned to the reference in Read 1. |
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Align 2 |
The percentage of clusters that aligned to the reference in Read 2. |
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PE Orientation |
The percentage of paired-end alignments with the expected orientation. |
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PE Resynthesis |
The ratio of first cycle intensities for Read 1 to first cycle intensities for Read 2. |
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PF |
The percentage of clusters passing filters. |
A cluster represents a clonal spot on the flow cell that contains the amplified DNA strands that will be sequenced.
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y-axis |
x-axis |
Description |
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Clusters |
Raw |
The total number of clusters detected in the run. |
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PF |
The total number of clusters passing filter in the run. |
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Unaligned |
The total number of clusters passing filter that did not align to the reference genome, if applicable. Clusters that are unindexed are not included in the unaligned count. |
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Unindexed |
The total number of clusters passing filter that were not associated with any index sequence in the run. |
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Duplicate |
The total number of clusters for a paired-end sequencing run which are considered to be PCR duplicates. |
Mismatch refers to any mismatch between sequence read and a reference genome after alignment.
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y-axis |
x-axis |
Description |
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Percent |
Cycle |
Plots the % mismatches for all clusters in a run versus cycle |