Local Run Manager Assembly Workflow
Module
Workflow Guide
Overview 3
Set Parameters 3
Analysis Methods 5
View Analysis Results 6
Analysis Report 6
Analysis Output Files 7
Technical Assistance 8
Document # 1000000057983 v00
June 2018
ILLUMINA PROPRIETARY
For Research Use Only. Not for use in diagnostic procedures.
This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for
the contractual use of its customer in connection with the use of the product(s) described herein and for no other
purpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwise
communicated, disclosed, or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina
does not convey any license under its patent, trademark, copyright, or common-law rights nor similar rights of any third
parties by this document.
The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in
order to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be
fully read and understood prior to using such product(s).
FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY
RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND DAMAGE
TO OTHER PROPERTY, AND WILL VOID ANY WARRANTY APPLICABLE TO THE PRODUCT(S).
ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S)
DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE).
© 2018 Illumina, Inc. All rights reserved.
All trademarks are the property of Illumina, Inc. or their respective owners. For specific trademark information, see
www.illumina.com/company/legal.html.
Document # 1000000057983 v00
For Research Use Only. Not for use in diagnostic procedures.
2
Local Run Manager Assembly Workflow Module Guide
Overview
The Assembly workflow assembles small genomes (< 20 Mb) either with or without the use of a reference
genome. It is best suited for the assembly of bacterial genomes, such as
E. coli
.
The Assembly workflow uses the Velvet software and writes assembly results in the FASTAfile format.
Compatible Library Types
The Assembly analysis module is compatible with specific library types represented by library kit categories
on the Create Run screen. For a current list of compatible library kits, see the Local Run Manager support
page on the Illumina website.
Input Requirements
The Assembly analysis module only requires the sequencing data files generated during a sequencing run.
About This Guide
This guide provides instructions for setting up run parameters for sequencing and analysis parameters for the
Assembly analysis module. For information about the Local Run Manager dashboard and system settings,
see the
Local Run Manager Software Guide (document # 1000000002702)
.
Set Parameters
1 If needed, log in to Local Run Manager.
2 Select Create Run, and select Assembly.
3 Enter a run name that identifies the run from sequencing through analysis.
The run name can contain alphanumeric characters, spaces, and the following special characters:
`~!@#$%-_{}.
4 [Optional] Enter a run description to identify the run.
The run description can contain alphanumeric characters, spaces, and the following special characters:
`~!@#$%-_{}.
Specify Run Settings
1 Select the library prep kit from the Library Prep Kit drop-down list.
2 Specify the number of index reads.
u 0 for a run with no indexing
u 1 for a single-indexed run
u 2 for a dual-indexed run
3 Select the read type for the run, if a change is possible.
4 Enter the number of cycles for the run.
5 [Optional] For Custom Primers, specify any custom primer information to be used for the run by selecting
the appropriate checkboxes.
Custom primer options vary based on your instrument or Local Run Manager implementation.
Document # 1000000057983 v00
For Research Use Only. Not for use in diagnostic procedures.
3
Local Run Manager Assembly Workflow Module Guide
Specify Module-Specific Settings
1 Enter a K-Mer Size.
This setting overrides the k-mer size used by Velvet. The default size is 31 and the minimum value is 3.
Odd-numbered values up to 255 are supported.
2 Select the On/Off toggle to enable or disable the following setting.
u Reverse Complement—(Only available with Nextera Mate Pair library prep kits) Off by default. When
enabled, all reads are reverse-complemented as they are written to FASTQ files.
Custom Analysis Settings
Custom analysis settings are intended for technically advanced users. If settings are applied incorrectly,
serious problems can occur.
Add a Custom Analysis Setting
1 From the Module-Specific Settings section of the Create Run screen, select Show Advanced Settings.
2 Select + Add custom setting.
3 In the custom setting field, enter the setting name as listed in the Available Analysis Settings section.
4 In the setting value field, enter the setting value.
5 To remove a setting, select .
Specify Samples for the Run
Specify samples for the run using the following options:
u Enter samples manually—Use the blank table at the bottom of the Create Run screen.
u Import sample sheet—Navigate to an external file in a comma-separated values (*.csv) format.
After you have populated the samples table, you can export the sample information to an external file. You
can use this file as a reference when preparing libraries or import the file when configuring another run.
Enter Samples Manually
1 Adjust the samples table to an appropriate number of rows.
u In the Rows field, use the up/down arrows or enter a number to specify the number of rows to add to
the table. Select to add the rows to the table.
u Select to delete a row.
u Right-click on a row in the table and use the commands in the contextual menu.
2 Enter a unique sample ID in the Sample ID field.
Use alphanumeric characters, dashes, or underscores.
3 Enter a sample name in the Sample Name field.
Use alphanumeric characters, dashes, or underscores. Spaces are not allowed in this field.
4 [Optional] Enter a sample description in the Sample Description field.
Use alphanumeric characters, dashes, underscores, or spaces.
5 If you have a plated kit, select an index plate well from the Index well drop-down list and skip to step8.
6 If applicable, specify an Index 1 sequence.
Document # 1000000057983 v00
For Research Use Only. Not for use in diagnostic procedures.
4
Local Run Manager Assembly Workflow Module Guide
Select Show Index Sequence/Show Index Names to toggle between showing the name of the index and
the index sequence.
7 If applicable, specify an Index 2 sequence.
NOTE
During analysis, the iSeq™ 100, MiniSeq™, and NextSeq™ Systems automatically reverse complement
the i5 indexes in custom library prep kits. Make sure that the i5 indexes are in the forward orientation.
8 If a reference is required, select a reference genome from the Genome Folder drop-down list.
9 [Optional] Enter a project name in the Sample Project field.
Use alphanumeric characters, dashes, or underscores. Spaces are not allowed in the Sample Project
field.
10 [Optional] Select Export Sample Sheet to export the sample information in *.csv format.
The exported sample sheet can be used as a template or imported when creating new runs.
11 Select Save Run.
Import Sample Sheet
1 If you do not have a sample sheet to import, see for instructions on how to create and export a sample
sheet. Edit the file as follows.
a Open the sample sheet in a text editor.
b Enter the sample information in the [Data] section of the file.
c Save the file. Make sure that the sample IDs are unique.
2 Select Import Sample Sheet at the top of the Create Run screen and browse to the location of the
sample sheet.
Make sure that the information in the sample sheet is correct. Incorrect information can impact the
sequencing run.
3 When finished, select Save Run.
Sample Sheet Fields
Manual editing of the sample sheet is intended for technically advanced users. If settings are applied
incorrectly, serious problems can occur.
Visit the Local Run Manager support page for available sample sheet settings. Settings must be entered as
specified to avoid analysis failure.
Analysis Methods
The Assembly workflow uses a
de Bruijn
graph methodology to assemble reads into contigs, which are
consensus DNA sequences representing overlapping sets of reads. The resulting contigs are written to a
FASTA file named contigs.fa in a subfolder of the Alignment folder named AssemblyN, where N is the sample
number.
Reads are randomly subsampled from the total data output to produce SN_L00#_Rx.fastq.gz files, where SN
refers to the sample number , # refers to the lane number, and x refers to the read number. These files
contain the reads used in the assembly process. The selection process is random but not stochastic,
meaning the same subset of reads are selected each time that the Assembly workflow is run. The
subsampling of reads prevents overloading of the RAM built into MiSeq instrument computer.
Document # 1000000057983 v00
For Research Use Only. Not for use in diagnostic procedures.
5
Local Run Manager Assembly Workflow Module Guide
If a reference genome is specified, the workflow performs the following steps:
u Compares contigs against the reference genome.
u Reorders contigs to match the order of the reference genome, as closely as possible.
u Generates the samples graph (dot-plot), which summarizes the match between contigs and the
reference genome. For more information, see
Samples Graph
on page 1.
NOTE
A reference genome is optional. Reference genomes can use either a *.fasta or *.fa file extension.
The assembly process uses the Velvet software. For a description of Velvet, see
Velvet: algorithms for de
novo short read assembly using de Bruijn graphs
, Zerbino and Birney, Genome Research 2008.
View Analysis Results
1 From the Local Run Manager dashboard, select the run name.
2 From the Run Overview tab, review the sequencing run metrics.
3 To change the analysis data file location for future requeues of the selected run, select the Edit icon,
and edit the output run folder file path.
The file path leading up to the output run folder is editable. The output run folder name cannot be
changed.
4 [Optional] Select the Copy to Clipboard icon to copy the output run folder file path.
5 Select the Sequencing Information tab to review run parameters and consumables information.
6 Select the Samples & Results tab to view the analysis report.
u If analysis was requeued, select the appropriate analysis from the Select Analysis drop-down list.
7 [Optional] Select the Copy to Clipboard icon to copy the Analysis Folder file path.
Analysis Report
Analysis results are summarized on the Samples & Results tab. The report is also available in a PDF file format
for each sample in the Analysis folder.
Sample Information
Column Description
Sample Name The sample name from the sample sheet.
Number of
Contigs
The total number of contigs identified in the sample.
N50 The length for which the collection of all contigs of that length, or longer, accumulates to half of the
total bases in the sample.
Minimum Contig
Length
The number of bases in the shortest contig in the sample.
Median Contig
Length
The median number of bases averaged over all contigs in the sample.
Mean Contig
Length
The mean number of bases averaged over all contigs in the sample.
Document # 1000000057983 v00
For Research Use Only. Not for use in diagnostic procedures.
6
Local Run Manager Assembly Workflow Module Guide
Column Description
Maximum Contig
Length
The number of bases in the longest contig in the sample.
Base Count The total number of bases in the sample.
Contig Plot Available when a reference genome is specified. Summarizes the match between contigs and the
reference genome.
Analysis Output Files
The following analysis output files are generated for the Assembly analysis module and provide analysis
results. Analysis output files are located in the Alignment folder.
File Name Description
Contigs.fa Contains the contigs for each assembly.
DotPlot.png File for the Contig Plot. Summarizes the match between contigs and the reference
genome.
Supplementary Output Files
The following output files provide supplementary information, or summarize run results and analysis errors.
Although these files are not required for assessing analysis results, they can be used for troubleshooting
purposes.
File Name Description
AdapterTrimming.txt Lists the number of trimmed bases and percentage of bases for each tile. This file
is present only if adapter trimming was specified for the run.
AnalysisLog.txt Processing log that describes every step that occurred during analysis of the
current run folder. This file does not contain error messages.
Located in the root level of the run folder.
AnalysisError.txt Processing log that lists any errors that occurred during analysis. This file will be
empty if no errors occurred.
Located in the root level of the run folder.
AssemblyRunStatistics.xml Contains summary statistics specific to the run.
Located in the root level folder.
CompletedJobInfo.xml Written after analysis is complete. Contains information about the run, such as
date, flow cell ID, software version, and other parameters.
Located in the root level of the run folder.
DemultiplexSummaryF1L1.txt Reports demultiplexing results in a table with one row per tile and one column per
sample.
Summary.xml Contains summary statistics specific to the run.
Contains a summary of mismatch rates and other base calling results.
Summary.htm Contains a summary web page generated from Summary.xml.
Document # 1000000057983 v00
For Research Use Only. Not for use in diagnostic procedures.
7
Local Run Manager Assembly Workflow Module Guide
Technical Assistance
For technical assistance, contact Illumina Technical Support.
Website:
www.illumina.com
Email:
techsupport@illumina.com
Illumina Customer Support Telephone Numbers
Region Toll Free Regional
North America
+1.800.809.4566
Australia
+1.800.775.688
Austria
+43 800006249 +43 19286540
Belgium
+32 80077160 +32 34002973
China
400.066.5835
Denmark
+45 80820183 +45 89871156
Finland
+358 800918363 +358 974790110
France
+33 805102193 +33 170770446
Germany
+49 8001014940 +49 8938035677
Hong Kong
800960230
Ireland
+353 1800936608 +353016950506
Italy
+39 800985513 +39 236003759
Japan
0800.111.5011
Netherlands
+31 8000222493 +31 207132960
New Zealand
0800.451.650
Norway
+47 800 16836 +47 21939693
Singapore
+1.800.579.2745
Spain
+34 911899417 +34 800300143
Sweden
+46 850619671 +46 200883979
Switzerland
+41 565800000 +41 800200442
Taiwan
00806651752
United Kingdom
+44 8000126019 +44 2073057197
Other countries
+44.1799.534000
Safety data sheets (SDSs)—Available on the Illumina website at support.illumina.com/sds.html.
Product documentation—Available for download in PDF from the Illumina website. Go to
support.illumina.com, select a product, then select Documentation & Literature.
Document # 1000000057983 v00
For Research Use Only. Not for use in diagnostic procedures.
8
Local Run Manager Assembly Workflow Module Guide
Illumina
5200 Illumina Way
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
techsupport@illumina.com
www.illumina.com
For Research Use Only. Not for use in diagnostic procedures.
© 2018 Illumina, Inc. All rights reserved.
Document # 1000000057983 v00
