1. GS Run Processor
:
1.4 Signal Processing Filters
: 1.4.1 Signal Filters and Corrections
1.4.1
Signal Filters and Corrections
Artifact filters and signal corrections are applied first, in the following order
1.
Screen out ghost wells (Amplicon pipelines only, wellScreener)
2.
Calculate and apply nucleotide normalization – normalizes the signal strengths of different base incorporations (nukeSignalStrengthBalancer).
3.
Calculate and apply inter-well crosstalk correction – corrects individual wells for the additional signal intensity conveyed by neighboring high intensity signal wells (blowByCorrector).
4.
Calculate and apply reagent flow event balancer – corrects anomalous signal spikes due to reagent valve events (valveEventBalancer).
5.
Calculate and apply out-of-phase error corrections (CAFIE - CArry Forward & Incomplete Extension – cafieCorrector)
•
Carry Forward occurs when a trace amount of nucleotide remains in a well after the apyrase wash, perpetuating premature nucleotide incorporations for specific sequence combinations during the next base flow. While this affects a small percentage of DNA strands per bead (~2%), it causes those strands to continue to incorporate nucleotides out-of-phase with respect to the rest of the strands and contributes to signal ‘noise’.
•
Incomplete Extension occurs when some DNA strands on a bead fail to incorporate during the appropriate base flow. This can be due to reactivity differences (it happens more often for ‘T’ flows, for example) and/or reagent and substrate local concentrations (it happens more for wells on the far end of the plate relative to the base flow direction). The strands that fail to incorporate must wait another flow cycle to continue sequencing and thus those strands will incorporate out-of-phase with the rest of the strands.
•
Recursive CAFIE uses an iterative approach to improve the CAFIE corrections. It is applied by default for all Runs on the GS Junior Instrument and for 400 cycle Runs on the GS FLX+ Instrument (using the GS FLX Titanium Sequencing Kit XL+).
6.
Re-calculate and re-apply nucleotide normalization – normalizes the signal strengths of different base incorporations (required only after standard CAFIE, nukeSignalStrengthBalancer).
7.
Calculate and apply signal droop correction – correct for signal reduction over the course of the sequencing Run exposure (individualWellScaler).
8.
Apply residual background subtraction and rescaling (mostLikelyErrorSubtractor).
9.
Calculate and apply well density correction – calculates the signal per base to filter out ghost wells (wellScreener).
The remaining corrected wells are considered to contain valid sequence information, albeit, of varying quality. They are counted as Raw Wells in the totalRawWells output metric.
