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NextSeq Sequencing

Run Monitoring

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By the end of this training, you will be able to:

– List the three options to monitor a run

– Describe the functionality of SAV

Session Objectives

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NextSeq Run Monitoring

Sequencing Screen in NCS

Monitor:

-On-instrument analysis

-Intensities Quality Scores

Sequencing Analysis Viewer

(SAV) in BaseSpace

Monitor run in greater detail

Sequencing Analysis Viewer

(SAV)

Monitor run in greater detail

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Viewing Run Metrics using

NextSeq Control Software

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NCS during On-Board Cluster Generation

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Note: Population of run metrics may be delayed by a couple of cycles due

to processing events

Run progress:• Status bar lists completed

cycles.

• Includes steps for

clustering, Read 1, Index 1

Read, Index 2 Read, Read

2, and the post-run wash

directly following the run.

A

B Q-Score:• Cycle 25

C Intensity: • Cycle 25

D Cluster Density (K/mm²):• Cycle 25

E Clusters Passing Filter:• Cycle 25

F Estimated Yield (Mb):• Cycle 25

G Flow Cell:• Shows which lane pair is

being imaged and on which

lane pair chemistry is being

performed.

Viewing Run Metrics on the Sequencing Screen

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Viewing Run Metrics using Sequencing

Analysis Viewer

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Sequencing Analysis Viewer

Files used by SAV

runinfo.xml

runparameters.xml

Interop files

Thumbnail files (optional)

Data Availability

Intensities after Cycle 25

Phasing/Prephasing after Cycle 25

Clusters Passing Filter (PF) after Cycle 25

Quality scoring after Cycle 25

PhiX alignment and error rates after Cycle 26

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SAV: Tabs

Run Metrics can also be reviewed offline from

any computer with access to the run folder

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SAV: Drop-down Options

Drop-down Options

11 COMPANY CONFIDENTIAL – INTERNAL USE ONLY

SAV: Summary

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SAV: Imaging Tab

13 COMPANY CONFIDENTIAL – INTERNAL USE ONLY

SAV: Indexing Tab

The Indexing tab is only available if the run is an index run

Note: For NextSeq data, indexing can be viewed in BaseSpace

after run completion or in SAV only after bcl2fastq2 conversion

14 COMPANY CONFIDENTIAL – INTERNAL USE ONLY

Viewing Run Metrics using Sequencing

Analysis Viewer in BaseSpace

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BaseSpace: Charts

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BaseSpace: Run Summary

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Summary of Run Monitoring

• Intensity, cluster density and Q scores are available during the run

Sequencing run metrics can be viewed on NCS during a run

• During a run and after a run has completed

• Provide more metric options than NCS

Sequencing run metrics can be viewed remotely using SAV at anytime

• Cycles 25 through run completion: Intensity, Cluster Density, % PF, Yield, Q scores

Run metrics are populated after cycle 25 in SAV during a run

• Trending of run metrics is highly recommended to identify outliers

Absolute or relative values for run metrics will be dependent on the type of library

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Online Training

• Sequencing Analysis Viewer (SAV)

• NextSeq 500: Does My Run Look Good?

Documentation

• Sequencing Analysis Viewer User Guide

NextSeq 500 Run Monitoring Resources

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Questions?