Metrics are updated as the run progresses. Select
Refresh at any time during the run to view updated
metrics. For more information, see the Sequencing
Analysis Viewer User Guide (part # 15020619).
The Sequencing Analysis Viewer is included in the
software installed on the instrument computer. You
can also install Sequencing Analysis Viewer on another
computer linked to the same network as the instrument to monitor run metrics remotely.
42. When the sequencing run is complete, the software initiates an
automatic postrun wash. The postrun wash uses wash solution
provided in the buffer cartridge and NaOCl provided in the
reagent cartridge. The automatic postrun wash takes approximately 90 min. When the wash is complete, the Home button
becomes active. Sequencing results remain visible on the screen
during the wash. After the wash, the sippers remain in the
down position to prevent air from entering the system. Leave
the cartridges in place until the next run.
43. Initial Analysis. As the run progresses, the control software
automatically transfers base call (BCL) files to BaseSpace
Sequence Hub, Local Run Manager, or another specified output location for secondary analysis.
The output BCL files are used for downstream analysis in
BaseSpace. Alternatively, use bcl2fastq conversion software for
FASTQ conversion and third-party analysis solutions. NextSeq
files require bcl2fastq v2.0, or later. For the latest version of
bcl2fastq, visit the NextSeq downloads page on the Illumina
website. The NextSeq 500 uses an implementation of RealTime Analysis (RTA) software called RTA2 (see Note 28).
3.8 Quality Control
of the Raw
Sequencing Data
3.8.1 Tools: FastQC,
Trimmomatic, and MultiQC
As mentioned above, the raw data generated from a sequencing run
come in the form of multiple .FASTQ files. These files encode the
sequence and the corresponding quality score for each base position of each read sequenced from the instrument. The most common way to quickly assess the quality of a .FASTQ read is using the
tool FastQC [21]. This program allows the user to quickly summarize the quality score of sequence data at each base position along
reads to determine whether any downstream processing such as
trimming the read length is needed. More information about the
quality score is available in Galaxy’s NGS tutorial (https://
galaxyproject.org/tutorials/ngs/). In the Galaxy analysis framework, the bioinformatic tools we recommend to perform quality
checks are FastQC, Trimmomatic [22], and MultiQC [23]. Additional information about FastQC and MultiQC may be found at
the following links: FastQC (https://www.babraham.ac.uk/projects/fastqc/), and MultiQC (https://multiqc.info/). A tutorial
for quality control is available as part of the available Galaxy training
504
Shuyi Ma et al.
Refresh at any time during the run to view updated
metrics. For more information, see the Sequencing
Analysis Viewer User Guide (part # 15020619).
The Sequencing Analysis Viewer is included in the
software installed on the instrument computer. You
can also install Sequencing Analysis Viewer on another
computer linked to the same network as the instrument to monitor run metrics remotely.
42. When the sequencing run is complete, the software initiates an
automatic postrun wash. The postrun wash uses wash solution
provided in the buffer cartridge and NaOCl provided in the
reagent cartridge. The automatic postrun wash takes approximately 90 min. When the wash is complete, the Home button
becomes active. Sequencing results remain visible on the screen
during the wash. After the wash, the sippers remain in the
down position to prevent air from entering the system. Leave
the cartridges in place until the next run.
43. Initial Analysis. As the run progresses, the control software
automatically transfers base call (BCL) files to BaseSpace
Sequence Hub, Local Run Manager, or another specified output location for secondary analysis.
The output BCL files are used for downstream analysis in
BaseSpace. Alternatively, use bcl2fastq conversion software for
FASTQ conversion and third-party analysis solutions. NextSeq
files require bcl2fastq v2.0, or later. For the latest version of
bcl2fastq, visit the NextSeq downloads page on the Illumina
website. The NextSeq 500 uses an implementation of RealTime Analysis (RTA) software called RTA2 (see Note 28).
3.8 Quality Control
of the Raw
Sequencing Data
3.8.1 Tools: FastQC,
Trimmomatic, and MultiQC
As mentioned above, the raw data generated from a sequencing run
come in the form of multiple .FASTQ files. These files encode the
sequence and the corresponding quality score for each base position of each read sequenced from the instrument. The most common way to quickly assess the quality of a .FASTQ read is using the
tool FastQC [21]. This program allows the user to quickly summarize the quality score of sequence data at each base position along
reads to determine whether any downstream processing such as
trimming the read length is needed. More information about the
quality score is available in Galaxy’s NGS tutorial (https://
galaxyproject.org/tutorials/ngs/). In the Galaxy analysis framework, the bioinformatic tools we recommend to perform quality
checks are FastQC, Trimmomatic [22], and MultiQC [23]. Additional information about FastQC and MultiQC may be found at
the following links: FastQC (https://www.babraham.ac.uk/projects/fastqc/), and MultiQC (https://multiqc.info/). A tutorial
for quality control is available as part of the available Galaxy training
504
Shuyi Ma et al.