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.
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 application note.
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