# Real Time Analysis (RTA3) on NovaSeq 6000 Overview

* **General Information**
  * Runs on the instrument Compute Engine (CE) and is 8x faster than previous versions.
  * Cannot resume if interrupted as tile images are stored in local system memory.
  * Uses rigid registration, 2-dye chemistry, aggregated .cbcls files.
  * Real-Time Analysis (RTA3) extracts intensities from images received from the camera, performs base calling, assigns a quality score to base calls, aligns to PhiX, and reports data in InterOp files for viewing in Sequencing Analysis Viewer (SAV).
* **What's New?**
  * Runs entirely on instrument CE and stores files to Z drive (temp folder location).
  * Algorithms rewritten to be entirely in C++.
  * Data stream optimization (vectorization and traffic flow optimizations).
  * Per cycle InterOp folder files.\
    \- Requires bcl2FASTQ2 v2.19 and later to parse.\
    \- BaseSpace Sequencing Hub has been updated to display NovaSeq InterOp folders.\
    \- For users who have their own homebrew SAV software, Open Source library to parse the new InterOp folder can be found [here](https://github.com/Illumina/interop).
  * Reduced number of reported Q scores.
* **Output** .cBCL (concatenated basecalls).
  * Aggregated by surface and lane (2 per lane per cycle).
  * Non passed filter (PF) clusters removed after cycle 25.
  * PhiX alignment is performed at cycle 26 on a subset of tiles for clusters that passed filter.
  * Filters out empty wells if detects 2 or fewer non-G calls in first 12 cycles.
  * Smaller due to reduced number of Q scores (2 base calls per byte before zipping).
* **Q scores**
* * Q40
  * Q30
  * Q20
  * Q10

RTA3 assigns each base call one of three quality scores based on the confidence of the base call. This Q-score reporting model reduces storage space and bandwidth requirements without affecting accuracy or performance.

For further information, see [NovaSeq 600 System Quality Scores and RTA3 Software](https://www.illumina.com/content/dam/illumina-marketing/documents/products/appnotes/novaseq-hiseq-q30-app-note-770-2017-010.pdf) application note.

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