> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/instrumentation/novaseq-6000/instrumentation-novaseq-6000-reference_material-list/000002465.md).
# Real-Time Analysis (RTA3) on NovaSeq 6000 Overview
**Background**
The NovaSeq 6000 runs RTA3, an implementation of Real-Time Analysis (RTA) software, on the instrument Compute Engine (CE). 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.
To optimize processing time, RTA3 stores information in memory. If RTA3 is terminated, processing does not resume and any run data being processed in memory is lost.
Additional information can be found in the Real-Time Analysis section of the [NovaSeq 6000 System Guide](https://emea.support.illumina.com/sequencing/sequencing_instruments/novaseq-6000/documentation.html).
**RTA3 Inputs**
RTA3 requires tile images contained in local system memory for processing. RTA3 receives run information and commands from the NovaSeq 6000 Control Software (NVCS).
**RTA3 Outputs**
Images for each color channel are passed in memory to RTA3 as tiles. From these images, RTA3 outputs a set of quality-scored base call files and filter files.
* Base call files: Each tile that is analyzed is included in a concatenated base call (\*.cbcl) file. Tiles from the same lane and surface are aggregated into 1 \*.cbcl file for each lane and surface.
* Filter files: Each tile produces a filter file (\*.filter) that specifies whether a cluster passes filters.
* Cluster location files: Cluster location (\*.locs) files contain the X,Y coordinates for every cluster in a tile. A cluster location file is generated for each run.
Output files are used for downstream analysis in BaseSpace Sequence Hub. Alternatively, use bcl2fastq2 Conversion software (v.2,19 or later) for FASTQ conversion and third-party analysis solutions.
RTA3 provides real-time metrics of run quality stored as InterOp files, which are a binary output containing tile, cycle, and read-level metrics. Viewing real-time metrics using Sequencing Analysis Viewer requires InterOp files. For the latest version of Sequencing Analysis Viewer, visit the [Sequencing Analysis Viewer downloads page](https://support.illumina.com/sequencing/sequencing_software/sequencing_analysis_viewer_sav/downloads.html) on the Illumina website. 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).
**RTA workflow**
1. Registration
* Registration aligns an image to the hexagonal array of nano wells on the patterned flow cell. Because of the ordered arrangement of nanowells, the X and Y coordinates for each cluster in a tile are predetermined.
* Cluster positions are written to a cluster location (s.locs) file for each run.
* If registration fails for any images in a cycle, no base calls are generated for that tile in that cycle. Use Sequencing Analysis Viewer to identify which images failed registration.
2. Intensity extraction
* After registration, intensity extraction calculates an intensity value for each nano well in a given image. If registration failed, the intensity for that tile cannot be extracted.
3. Phasing correction
* During the sequencing reaction, each DNA strand in a cluster extends by one base per cycle. Phasing and prephasing occur when a strand becomes out of phase with the current incorporation cycle.
* Phasing occurs when a base falls behind.
* Prephasing occurs when a base jumps ahead.
* RTA3 corrects the effects of phasing and prephasing, which maximizes the data quality at every cycle throughout the run.
4. Base calling
* Base calling determines a base (A, C, G, or T) for every cluster of a given tile at a specific cycle. The NovaSeq 6000 uses two-channel sequencing, which requires only two images to encode the data for four DNA bases, one from the red channel and one from the green channel.
* A no call is identified as N. No calls occur when a cluster does not pass filter, registration fails, or a cluster is shifted off the image.
* Clusters Passing filter
* During the run, RTA3 filters raw data to remove reads that do not meet the data quality threshold. Overlapping and low-quality clusters are removed.
* For two-channel analysis, RTA3 uses a population-based system to determine the chastity (intensity purity measurement) of a base call. Clusters pass filter (PF) when no more than one base call in the first 25 cycles has a chastity below a fixed threshold.
* PhiX alignment is performed at cycle 26 on a subset of tiles for clusters that passed filter. Clusters that do not pass filter are not base called and not aligned.
5. Quality scoring
* A quality score (Q-score) is a prediction of the probability of an incorrect base call. A higher Q-score implies that a base call is higher quality and more likely to be correct. After the Q-score is determined, results are recorded in base call (\*.cbcl) files.
* 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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