Sequencing Analysis Viewer User Guide 15020619 F
Sequencing Analysis Viewer User Guide 15020619 F
Sequencing Analysis Viewer User Guide 15020619 F
User Guide
Revision History 3
Introduction 4
Setting Up Sequencing Analysis Viewer Software 5
Data Availability 8
Loading Data 9
Analysis Tab 10
Imaging Tab 17
Summary Tab 19
Tile Status Tab 21
TruSeq Controls Tab 22
Indexing Tab 26
InterOp Files 28
Technical Assistance
ILLUMINA PROPRIETARY
Part # 15020619 Rev F
October 2014
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© 2014 Illumina, Inc. All rights reserved.
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SeqMonitor, SureMDA, TruGenome, TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, verifi, VeriSeq, the
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Revision History
Part # Revision Date Description of Change
15020619 F October Added support for HiSeq X and InterOp files for
2014 RTA 1.1x and 2.x.
15020619 E January Added support for NextSeq, and descriptions of
2014 InterOp files.
15020619 D January Added figure of in-line controls in a successful TruSeq
2013 DNA PCR-Free run
15020619 C October User Guide for Sequencing Analysis Viewer
2012 Software v1.8.20 and up, describes binned Q-scores.
Requirements
Sequencing Analysis Viewer Software does not need an advanced personal computer,
because the instrument control computer running the Real-Time Analysis (RTA) software
does the heavy computational work. The following items are required to run the
software:
} Desktop computer running 32-bit or 64-bit version Windows XP, Windows Vista, or
Windows 7
} Network access to the run data
} .Net framework 4.0
NOTE
The Run Folder screen varies depending on your Illumina sequencing system and the
operating system on your personal computer.
3 Click Refresh.
The software starts loading data, and when completed it shows available quality
metrics for that run.
If the SAV Software does not display metrics, check whether you pointed the software to
a valid run folder, and whether you have the proper permissions to view the data. If
there is no valid run folder, the software shows an error message.
FWHM 1 1 1 1 1
Template 4 7 5 4 1
Generation
Intensity 4 7 5 5 1
Corrected Intensity 12 12 5 12 3
Clusters Passing 25 25 25 25 25
Filter
Called Intensity 25 25 5 25 3
% Base 25 25 25 25 25
% Phasing, % 25 25 25 25 25
Prephasing
% Aligned 25 25 25 25 25
Error Rate 25 25 25 25 25
% PF 25 25 25 25 25
} Corrected intensities are generated after phasing and prephasing are calculated. Base
calls are generated after corrected intensities are generated.
} MiSeq, NextSeq, and HiSeq instruments running HCS 2.2.38 or higher generate
phasing and prephasing estimates empirically for every cycle.
TIP
In any screen, click the Refresh button to gather the latest metrics.
} The phasing reported in the SAV Software is the tile median slope of the observed
phasing corrections for cycles 1–25.
NOTE
The Run Folder screen varies depending on your Illumina sequencing system and the
operating system on your personal computer.
3 Click Refresh.
The SAV Software starts loading data showing quality metrics for that run.
NOTE
If you are using the SAV Software to view metrics from the MiSeq sequencing instrument
(which has only one lane), select 1 or All in the Lane drop-down list.
Status Pane
The Status pane reports the progress of the analysis. The following steps are reported:
} Extracted: the last complete cycle for which image analysis has been performed.
} Called: the last complete cycle that has been base-called.
} Scored: the last complete cycle that has been quality scored.
The statistics are updated as soon as there is new analysis information available, and
you hit the Refresh button.
You can monitor the following quality metrics with this chart:
} Intensity—This chart shows the intensity by color and cycle of the 90% percentile of
the data for each tile. On platforms using two-channel sequencing, only red and
green are shown. On platforms using four-channel sequencing, 4 channels are
shown.
} FWHM—The average full width of clusters at half maximum (in pixels). For a more
detailed description, see en.wikipedia.org/wiki/Full_width_at_half_maximum.
} Corrected Intensity:
• MiSeq and HiSeq: The intensity corrected for cross talk between the color
channels and phasing and prephasing.
You can monitor the following quality metrics with this plot:
} Intensity—This plot shows the intensity by color of the 90% percentile of the data for
each cycle.
} FWHM—The average full width of clusters at half maximum (in pixels). For a more
detailed description, see en.wikipedia.org/wiki/Full_width_at_half_maximum.
} Corrected Intensity:
• MiSeq and HiSeq: The intensity corrected for cross talk between the color
channels and phasing and prephasing.
• NextSeq: Intensities in NextSeq two-channel sequencing are calculated using a
different method than in four-channel sequencing. The values presented here are
not used in base calling, but can aide in assessing progress of a run. For
NextSeq, called and corrected intensities graphs are identical.
} Called Intensity—The intensity for the called base.
} % Base—The percentage of clusters for which the selected base has been called.
} Signal to Noise—The signal to noise ratio is calculated as mean called intensity
divided by standard deviation of non-called intensities. Not calculated for NextSeq
two-channel sequencing or HiSeq X.
} Error Rate—The calculated error rate, as determined by a spiked in PhiX control
sample. If a PhiX control sample is not run in the lane, this chart is not available.
} % Perfect Reads—The percentage of reads that align perfectly, as determined by a
spiked in PhiX control sample. If a PhiX control sample is not run in the lane, this
chart is not available. Not calculated for NextSeq two-channel sequencing.
} %Q > 20, %Q > 30—The percentage of bases with a quality score of 20 or 30 or
higher, respectively. These charts are generated after the 25th cycle, and the values
represent the current cycle. Click the Accum checkbox to view all cumulative data up
to that point. Leave the box unchecked to display only the current cycle.
You can monitor the following quality metrics with this plot (blue boxes are for raw
clusters, green boxes for clusters passing filter):
} The density of clusters for each tile (in thousands per mm2).
} The number of clusters for each tile (in millions).
} % Phasing, % Prephasing—The percentage of molecules in a cluster for which
sequencing falls behind (phasing) or jumps ahead (prephasing) the current cycle
within a read. The graphs are split out by read.
For MiSeq, NextSeq, and HiSeq X, RTA generates phasing and prephasing estimates
empirically for every cycle. The value displayed here is therefore not used in the
actual phasing/prephasing calculations, but is an aggregate value determined from
the first 25 cycles. For most applications, the reported value should be very close to
the applied value. However, on MiSeq or NextSeq, for low diversity samples or
samples with unbalanced base composition, the reported value might not accurately
reflect the values being applied because the value changes from cycle to cycle. HiSeq
X currently does not support low diversity samples or samples with unbalanced
base composition.
} % Aligned—The percentage of the sample that aligned to the PhiX genome. The
graphs are split out by read.
A Cutoff Slider
The Q-score is based on the Phred scale. The following table lists Q-scores and the
corresponding estimated base call error rate at that Q-score.
Yield Total The number of bases sequenced, which is updated as the run
progresses.
Projected Total Yield The projected number of bases expected to be sequenced at
the end of the run.
Yield Perfect The number of bases in reads that align perfectly, as
determined by alignment to PhiX of reads derived from a
spiked in PhiX control sample. If a PhiX control sample is not
run in the lane, this chart is not available.
Yield <=3 errors The number of bases in reads that align with three errors or
less, as determined by a spiked in PhiX control sample. If a
PhiX control sample is not run in the lane, this chart is not
available, and shows a zero value. This value is not calculated
for NextSeq two-channel sequencing, and the value shown is
always zero.
Aligned The percentage of the sample that aligned to the PhiX
genome, which is determined for each level or read
independently.
% Perfect [Num Usable Cycles] The percentage of bases in reads that align perfectly, as
determined by a spiked in PhiX control sample, at the cycle
indicated in the brackets. If no PhiX control sample is run in
the lane, this chart shows 0% and the number of cycles used.
This value is not calculated for NextSeq two-channel
sequencing, and the value shown is always zero.
% <=3 errors [Num Usable The percentage of bases in reads that align with three errors
Cycles] or less, as determined by a spiked in PhiX control sample, at
the indicated cycle. If no PhiX control sample is run in the
lane, this chart shows 0% and the number of cycles used. This
value is not calculated for NextSeq two-channel sequencing,
and the value shown is always zero.
Error Rate The calculated error rate of the reads that aligned to PhiX.
Intensity Cycle 1 The average of the A channel intensity measured at the first
cycle averaged over filtered clusters.
% Intensity Cycle 20 The corresponding intensity statistic at cycle 20 as a
percentage of that value at the first cycle. 100%x(Intensity at
cycle 20)/(Intensity at cycle 1).
%Q>=30 The percentage of bases with a quality score of 30 or higher,
th
respectively. This chart is generated after the 25 cycle, and
the values represent the current cycle.
The following metrics are available in the Read tables, split out by lane:
The bottom of the page contains two buttons, Copy to Clipboard and Generate IVC Plots,
so you can copy the data to your computer, and generate your IVC plots at any time
during the run.
In every tile, two numbers indicate the progress of base calling and quality scoring. For
every tile, the top number indicates the last cycle that has been base called, and the
bottom number indicates the last cycle for which quality has been scored.
Figure 14 Base Calling and Quality Scoring Progress
A successful run should have a positive signal for all four controls at one or two
adjacent sizes. The reported size should correspond to the excised fragment. The absolute
number of reads for each control does not matter much, but there should be a clear peak
near the expected size. An example of a successful run for a TruSeq DNA 350 bp gel
excised fragment is shown here.
Figure 15 In-Line Controls in a Successful Run
Libraries prepared with the TruSeq DNA PCR-Free Sample Prep kit show a narrow size
distribution for CTE1 and CTE2. However, CTA and CTL size distributions are broad as
the size selection step occurs upstream of the addition of these controls. An example of a
successful run with a 350 bp size-selected insert is shown here.
If a run was unsuccessful due to an inefficient step in the sample prep, the controls
would look like one of the following examples:
This information is also displayed in graphical form. In the graphical display, indexes
are ordered according to the unique Index Number assigned by AS.
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