Does my sequencing run look good?

Overall sequencing run performance is evaluated by determining whether the sequencing run meets the Illumina specifications for quality scores and data output. Actual run performance will vary based on sample type, quality, and clusters passing filter. Specifications are based on the Illumina PhiX control library at supported cluster densities.

Where can I find instrument specifications?

Follow the links below to the instrument specification pages:

iSeq 100 | MiniSeq | MiSeq | NextSeq 500/550 | NextSeq 1000/2000 | HiSeq 3000/4000 | NovaSeq 6000 | NovaSeq X/X Plus

The Sequencing Analysis Viewer (SAV) is a free software used to assess the performance of sequencing runs, and can be downloaded from the Illumina website:

• SAV v2.4.7 of SAV on all instruments except on-instrument MiSeq and NextSeq1000/2000; SAV v2.4.7 is compatible for all off-instrument (remote) use running Windows 7 or later

• SAV v1.8.37 of SAV for on-instrument MiSeq viewingOnce SAV is installed, open it and select the tab containing the desired query information.

How do I determine if my run meets spec?

Below is an example of a PhiX validation run (2 x 151 bp) on the MiSeq, using v2 reagents. The specifications for this run are as follows:

• Total data output of 4.5-5.1 gigabases (Gb)

• At least 80% of bases called with a quality score of 30 or higher (at least 80% ≥ Q30)

To determine the quality score, review the Analysis tab Q score Distribution chart and the Summary tab as shown below.

Analysis Tab:

Summary Tab:

The quality specification for a MiSeq paired-end 151-cycle run is Q30 ≥ 80%. The run meets this specification, as the percent ≥ Q30 is >94%.

To determine the yield of the run, review the information in the Summary tab as shown below.

The yield specification for a paired-end 151-cycle run is >4.4 Gb. The run meets this specification, as the total yield is 6.10 Gb.

1. Does the overall quality (Q30) score meet specification?  
2. Does the total data output meet specification?**What additional information can I obtain from SAV?**  

The following images are from BaseSpace public data set: “MiSeq: Nextera DNA Flex (replicates of E. coli, B. cereus, and R. sphaeroides)”. Note: Nextera DNA Flex has been renamed to Illumina DNA Prep.

Analysis Tab: Overview of the run metrics.

1. Flow Cell Chart shows color-coded metrics per tile for the entire flow cell.  
2. Data by Cycle displays various metrics for each cycle of the run. Select the displayed metric, lane, surface, and channel using the drop-down lists.  
3. Q Score Distribution shows a quick overview of the quality of the run. The Q30 for the whole run is found in the upper right of this box.  
4. Data by Lane shows plots of metrics per lane.  
5. Q score Heatmap displays a heat map for Q score by cycle.    

Imaging Tab: Displays thumbnails from the run if available.

1. Toggle which base or color channel image to view here.  
2. If thumbnails are saved for the run, they are displayed here.    

Summary Tab: Provides basic data quality metrics summarized per lane and per read.

1. Run summary per read, including quality, is reported here.  
2. More details per read including the exact density, clusters Passing Filter (PF), and % aligned.    

Indexing Tab: Total and Per Sample % Reads Identified if a sample sheet was used and demultiplexing was performed.

1. Metrics for the entire run. Note: % Reads Identified will not include any PhiX because PhiX is not indexed.  
2. Metrics per sample, using indexing information from the sample sheet.  
3. Graph plotting the percent of reads identified for each sample present in the sample sheet.    

Additional Resources:

• Sequencing Analysis Viewer Software Guide

• Sequencing Analysis Viewer (SAV) Training

• Plotting % Occupied by % Pass Filter to optimize loading concentration for NovaSeq 6000 and iSeq 100 platforms

For any feedback or questions regarding this article (Illumina Knowledge Article #1922), contact Illumina Technical Support [e-mail].