SMRT® Analysis Barcoding Overview (v9.0)
Introduction

This document applies to Sequel® and Sequel II Systems using SMRT®
Link v9.0.
Note: For information on SMRT Link v8.0, see the document SMRT
Analysis Barcoding Overview v8.0, available here.
SMRT Link v9.0 includes a streamlined end-to-end barcoding workflow for
automated analysis of multiplexed samples. It enables users to definebarcodes and their associated sample names for an experiment at Run
Design.
This workflow provides extensive QC metrics for evaluation of barcoding
performance.
The SMRT Link graphical user interface supports up to 10,000 barcodes
per sample. Support for more than 10,000 samples is available using thecommand-line. See the document SMRT® Tools Reference Guide (v9.0),
available here.
This document covers:• Barcoding designs, strategies and modes for preparing barcodedsamples and data analysis for all the applications available with SMRT
Link v9.0.
• Identifying barcodes using SMRT Link.
• Finding demultiplexed subread.bam files.
This document does not include information about barcoding designs andanalysis for the Iso-Seq® and No-Amp application. For details aboutbarcoding samples for Iso-Seq analysis, see the document SMRT Link
User Guide (v9.0), available here.

PacBioBarcoding
Background

Page 1

Barcoding
Strategies

There are several options for barcoding samples for SMRT sequencing:
1. Barcoded Primers– Target-specific primers are tailed with 16-bp barcodes. Use thismethod if your primers are already fully validated.
2. Barcoded Universal Primers– This method requires two rounds of PCR. The first round uses
target-specific primers tailed with universal forward and reverse
sequences.
– For the second PCR round, round one PCR products are barcodedusing F/R universal primers tailed with 16-bp barcodes.
– For symmetric barcoding, PacBio recommends using ourcommercially-available 96 Barcoded Universal Primers kit. For
asymmetric barcoding, we recommend using the barcoded M13
Primer approach.
3. Barcoded Overhang Adapters– PacBio barcodes are added to the sample through ligation of
barcoded overhang SMRTbell® adapters.
– Recommended for validated PCR systems and smaller projects.
– Besides amplicons, barcoded overhang adapters are also used for
microbial multiplexing.
4. Linear Barcoded Adapters for Probe-Based Captures– Recommended for probe-based captures of genomic regions ofinterest.

All workflows include barcodes to uniquely identify samples, but theworkflows differ in how the barcodes are incorporated into the SMRTbell
template.

Page 2

Description of Linear Barcoded Adapters for Multiplexing Target
Capture Experiments
For targeted captures only, use Linear Barcoded Adapters:

Linear barcoded adapters containing universal sequences are ligated to
double-stranded DNA fragments for amplification and capture. This
workflow supports up to 24 barcodes, which can be ordered from any oligo
synthesis provider.
Demultiplexing of such experiments is supported through SMRT Link
command-line options.

BarcodeScoring Modes

The demultiplexing algorithm included in SMRT Link v9.0 only requires
information about the barcoding sequences – whether they are the sameor different on either side of the read. Barcode orientation does not need
to be specified. The demultiplexing algorithm scores all barcode
sequences in both orientations - forward and reverse complement.
This section describes barcode scoring modes and provides information
about the different experiment options depending on barcoding strategy.

The Demultiplex Barcodes application in SMRT Link supports
demultiplexing of subreads and CCS data. The following terminology isbased on the per (sub-) read view.

Page 3

Symmetric Mode
For symmetric and tailed library designs, the same barcode is attached
to both sides of the insert sequence of interest. The only difference is theorientation of the trailing barcode. For barcode identification, one read witha single barcode region is sufficient. Symmetric barcoding is used forsamples constructed using Barcoded Overhang Adapters, Barcoded
Universal Primer and target enrichment (linear). This is also the default
scoring mode in SMRT Link v9.0 and later.

Asymmetric Mode
Barcode sequences are different on the ends of the SMRTbell Template.
Asymmetric mode is used with the M13 barcoding procedure. Pacific
Biosciences recommends using this mode only for small inserts (up to 5kb) where both ends of the insert are expected to be sequenced. Bothbarcodes must be detected.
Note: For both Symmetric and Asymmetric modes, the limit for unique
individual barcode sequences is 768, and the limit for the number ofdifferent barcode pairs is 15,000.
When running the Demultiplex Barcodes applications in SMRT Link, set
the Same Barcodes on Both Ends of the Sequence option to Off.

Mixed Mode
Libraries with combined symmetric and asymmetric barcoding are notsupported.

Which
Barcoding
Strategies toUse

For application recommendations, see Overview Sequel Systems
Application Options and Sequencing Recommendations.
Note: For multiplexing microbial genomes experiments, click here to see
details about the workflows, analysis and barcoding kits that we provide.
The following table includes the names of FASTA files used for analysis:

1 Using Barcoded Overhang Adapter Kit 8A (PN 101-628-400) and Barcoded

Overhang Adapter Kit 8B. (PN 101-628-500)

2 Using oligos from PacBio recommended lists.

Page 4Custom
Symmetric
Barcode FASTA

Independent of the barcoding strategy and mode, if you are using custom
barcodes, you must generate a FASTA file of barcode sequences. This fileshould:• Include one entry per barcode sequence.
• Include no duplicate sequences.
• Be orientation-agnostic (forward or reverse-complement, but notreversed.)

Please name your barcodes with an alphabetic character prefix to avoid
later confusion of barcode name and index. Duplicate names are notallowed.

Working with
Barcoded Data
in SMRT Link
v9.0

This section describes how to use SMRT Link to work with barcoded data.
Demultiplex Barcodes analysis is powered by the Lima SMRT Analysis
tool.
The canned data provided with SMRT Link v9.0 includes 7 barcode sets:• gDNA_Sample_Amplification_Adapter• IsoSeq_Primers_12_Barcodes_v1• IsoSeqPrimers_v2 (Includes the content of IsoSeqPrimers as wellas support for NEB and Clontech primers.)• Sequel_16_barcodes_v1• Sequel_16_barcodes_v3• Sequel_96_barcodes_v1• Sequel_384_barcodes_v1

Step 1: Specify the Barcode Setup and Sample Names in a Run
Design1. In SMRT Link, create a new Run Design as described in “Creating a
New Run Design” on page 11 of the SMRT Link User Guide (v9.0).
Before you finish the new Run Design, perform the following steps.

Page 5

2. Click Barcoded Sample Options and then click Yes for Sample is
Barcoded. Additional fields related to barcoding display.
3. Specify a Barcode Set using the dropdown list.
Note: You can specify up to 15,000 samples. Specifying more than
10,000 samples may cause a delay of several minutes in analysis
submission.
4. Specify if the same barcodes are used on both ends of the sequences.
– Selecting Yes specifies symmetric and tailed designs where all thereads have the same barcodes on both ends of the insert sequence.
Barcode analysis of such experiments retains only data with thesame barcode identified on both ends.
– Selecting No specifies asymmetric designs where the barcodes aredifferent on each end of the insert. Barcode analysis of such
experiments retains any barcode pair combination identified in the
Data Set.
5. SMRT Link automatically creates a CSV-format Autofilled Barcode
Name File. The barcode name is populated based on your choice ofbarcode set, and if the barcodes are the same at both ends of thesequence. The file includes a column of automatically-generated Bio
Sample Names 1 through N, corresponding to barcodes 1 through N,
for the biological sample names.
– To specify the biological sample names corresponding to eachbarcode, click Download Name File, enter the biological sample
names associated with the barcodes in the second column, andsave the file. (Use alphanumeric characters, spaces, hyphens,
underscores, colons, or periods only - other characters will be
removed automatically, with a maximum of 40 characters.) If you
did not use all barcodes in the Autofilled Barcode Name file in thesequencing run, delete unused rows.
6. Select the Barcoded Sample Name File you edited in Step 5.
7. Click Save.

Page 6

Step 2: Perform the Sequencing Run
Load the samples and perform the sequencing run, using the Run Design
you created in Step 1. The demultiplexing analysis is performed
automatically on the SMRT Link Server once the data is transferred fromthe Sequel/Sequel II System. This creates an analysis of type
Demultiplex Barcodes (Auto) in the SMRT Analysis module. You canclick to select this analysis and review the reports and data created. If
everything looks fine, you can continue to Step 4 and use thedemultiplexed Data Set(s) created by the run as input to further analysis.
Note: By default, Demultiplex Barcodes (Auto) creates one Data Set
per auto-detected barcode within the selected barcode set. It also applies
a Data Set filter of a minimum barcode score greater than 26 for optimal
results in secondary analyses. If used, the analysis parameter Filters to
add to the Data Set overrides other barcode filtering, even if the barcodescore set with it is lower than 26.

Step 3: (Optional) Run the Demultiplex Barcodes Application
If you did not specify the barcode setup in the Run Design, or if you needto change any of the parameters used in the Demultiplex Barcodes
analysis automatically launched from Run Design, run the Demultiplex
Barcodes application. This application separates reads by barcode andcreates a new demultiplexed Data Set that you can then use as input toother secondary analysis applications.
1. Click + Create New Analysis.
2. Enter a name for the analysis.
3. Select the type of data to use for the analysis:– Sequel Data: Subreads from Sequel or Sequel II Systems.
– CCS Data: Single Molecule consensus reads generated from the
CCS analysis.
The Data Sets box displays the appropriate Data Sets available for the
analysis.
4. In the Data Sets box, select one or more Data Sets to be analyzed
together.
5. Click Next.
6. Select Demultiplex Barcodes from the Applications list.

Page 7

7. Specify a Barcode Set (barcode sequence file.)
Note: You can specify up to 15,000 samples. Specifying more than
10,000 samples may cause a delay of several minutes in analysis
submission.
8. Specify the name for the new demultiplexed Data Set that will display
in SMRT Link.
9. Specify if the same barcodes are used on both ends of the sequences.
– Selecting On specifies symmetric and tailed designs where all thereads have the same barcodes on both ends of the insert sequence.
Barcode analysis of such experiments retains only data with thesame barcode identified on both ends.
– Selecting Off specifies asymmetric designs where the barcodes aredifferent on each end of the insert. Barcode analysis of such dataretains any barcode pair combination identified in the Data Set.
10. Specify the Minimum Barcode Score: Reads with barcode scores
below the value are not included in the analysis. We recommend thatyou set this value to 26 for all applications.
11. SMRT Link automatically creates a CSV-format Autofilled Barcode
Name File. The barcode name is populated based on your choice ofbarcode set, and if the barcodes are the same at both ends of thesequence. The file includes a column of automatically-generated Bio
Sample Names 1 through N, corresponding to barcodes 1 through N,
for the biological sample names.
– To specify the biological sample names corresponding to eachbarcode, click Download Name File, enter the biological sample
names associated with the barcodes in the second column, andsave the file. (Use alphanumeric characters, spaces, hyphens,
underscores, colons, or periods only - other characters will be
removed automatically, with a maximum of 40 characters.) If you
did not use all barcodes in the Autofilled Barcode Name file in thesequencing run, delete those rows.

Page 8

– Note: Open the CSV file in a text editor and check that the columns
are separated by commas, not semicolons or tabs.
12. Select the Barcoded Sample Name File you edited in Step 11.
13. (Optional) Specify any advanced parameters.
14. Click Start. After the analysis is finished, a new demultiplexed Data
Set is available.
Note: For information about the reports generated by the Demultiplex
Barcodes application, see “Demultiplex Barcodes Reports and Data Files” on page
11.

Step 4: Run Applications Using the Demultiplexed Data as
Input
All secondary analysis applications except Demultiplex Barcodes canuse demultiplexed Data Sets as input.
Note: For Iso-Seq analysis with barcoded samples, use the Iso-Seq
application instead of the Demultiplex Barcodes application, as the IsoSeq application already includes the demultiplexing step as part of thepipeline. When performing multiplexed Iso-Seq analysis, ensure that the
Run Design Sample Is Barcoded option is set to No (the default setting).
Then, in SMRT Analysis, go straight to the Iso-Seq application and, in theparameters section, select a Primer Set containing multiple primers, suchas IsoSeq_Primers_12_Barcodes_v1.
1. Select the secondary analysis application to use.
2. Click the number in the Demultiplexed Subsets column, then selectthe demultiplexed Data Set to use as input:– You can select the entire Data Set as input, or one or more specificoutputs from selected barcodes, to a maximum of 16 sub-Data Sets,
12 for Iso-Seq Analysis.
3. Additional Analysis Type options become available. You can selectfrom the following options:Page 9

– One Analysis for All Data Sets: Runs one analysis using all theselected barcode Data Sets as input, for a maximum of 30 Data
Sets.
– One Analysis per Data Set - Identical Parameters: Runs one
separate analysis for each of the selected barcode Data Sets, using
the same parameters, for a maximum of 10,000 Data Sets.
Optionally click Advanced Parameters and modify parameters.
– One Analysis per Data Set - Custom Parameters: Runs one
separate analysis for each of the selected barcode Data Sets, using
different parameters for each Data Set, for a maximum of 16 Data
Sets. Click Advanced Parameters and modify parameters. Thenclick Start and Create Next. You can then specify parameters foreach of the included barcode Data Sets.
– Note: The number of Data Sets listed is based on testing using
PacBio's suggested compute configuration, listed in SMRT Link
Software Installation (v9.0).
4. Click Start to submit the analysis.

Demultiplex
Barcodes Input
and Output

Given an input set of barcodes and a BAM Data Set, the Demultiplex
Barcodes application produces:• A set of BAM files whose reads are annotated with the barcodes;• A subreadset file that contains the file paths of that collection of
barcode-tagged BAM files and their related files.
Barcode Set (Required):• Specify a barcode sequence file to separate the reads.
New Data Set Name (Required):• Specify the name for the new demultiplexed Data Set that will display
in SMRT Link.
Same Barcodes on Both Ends of Sequence (Default = ON)• Specify On to retain all the reads with the same barcodes on bothends of the insert sequence, such as symmetric and tailed designs.
(See “Barcode Scoring Modes” on page 3 for information on barcode
designs.)• Specify Off to specify asymmetric designs where the barcodes aredifferent on each end of the insert.

Page 10

Minimum Barcode Score (Default = 0)
• A barcode score measures the alignment between a barcode
attached to a read and an ideal barcode sequence, and is an indicatorof how well the chosen barcode pair matches. It ranges between 0 (nomatch) and 100 (a perfect match). Specifies that reads with barcodescores below this minimum value are not included in analysis.
Autofilled Barcode Name File
• SMRT Link automatically creates a CSV-format Autofilled Barcode
Name File. The barcode name is populated based on your choice ofbarcode set, and if the barcodes are the same at both ends of thesequence. The file includes a column of automatically-generated Bio
Sample Names 1 through N, corresponding to barcodes 1 through N,
for the biological sample names.
• To specify the biological sample names corresponding to eachbarcode, click Download Name File, enter the biological sample
names associated with the barcodes in the second column, and savethe file. (Use alphanumeric characters, spaces, hyphens, underscores,
colons, or periods only - other characters will be removed
automatically, with a maximum of 40 characters.) If you did not use
all barcodes in the Autofilled Barcode Name file in the sequencing run,delete those rows.
• Note: Open the CSV file in a text editor and check that the columns are
separated by commas, not semicolons or tabs.
Barcoded Sample File Name
• Select the Barcoded Sample Name File you edited previously.

Demultiplex
BarcodesReports andData Files

Reports and Data FilesThe Demultiplex Barcodes application generates the following reports:
Barcodes > Summary Metrics
•
•
•
•
•
•
•

Unique Barcodes: The number of unique barcodes in the sequence data.
Barcoded Reads: The number of barcoded reads in the sequence data.
Mean Reads: The mean number of reads per barcode.
Max. Reads: The maximum number of reads per barcode.
Min. Reads: The minimum number of reads per barcode.
Mean Read Length: The mean read length of reads per barcode.
Mean Longest Subread Length: The mean length of the longest subread ineach barcoded sample.
• Unbarcoded Reads: The number of reads without barcodes in the sequence
data.

Barcodes > Barcode Data
• Bio Sample Name: The name of the biological sample associated with thebarcode.
• Barcode Index: The index number associated with the barcode.
• Barcode Name: A string containing the pair of barcode indices for which thefollowing metrics apply.
• Polymerase Reads: The number of polymerase reads associated with thebarcode.

Page 11• Subreads: The number of subreads associated with the barcode.
• Bases: The number of bases associated with the barcode.
• Mean Read Length: The mean read length of reads associated with thebarcode.
• Longest Subread Length: The longest subread length associated with thebarcode.
• Mean Barcode Quality: The mean barcode quality associated with thebarcode.
• Rank Order (Num. Reads): The rank order of this barcode in terms of thenumber of reads.

Barcodes > Barcoded Read Statistics• Number of Reads per Barcode: Line graph displays the number of sorted
reads per barcode.
– Good performance: The Number of Reads per Barcode line (blue) should
be mostly linear. Note that this depends on the choice of Y-axis scale. The
mean Number of Reads per Barcode line (red) should be near the middle of
the graph and should not be skewed by samples with too many or too few
barcodes.
– Questionable performance: A sharp discontinuity in the blue line,
followed by no yield, with the red line way far from the center. Check theoutput file Inferred Barcodes, note the correct barcodes used, andconsider reanalyzing the multiplexed samples with the correct Bio Sample
names for the barcodes actually used. If you reanalyze the data, ensurethat the Barcode Name File includes only the correct barcodes used.
• Barcode Frequency Distribution: Histogram distribution of read counts per
barcode.
– Good performance: A uniform distribution, which is most often a fairly tightsymmetric normal distribution, with few barcodes in the tails.
– Questionable performance: A large peak at zero. This can indicate use ofincorrect barcodes. Check the output file Inferred Barcodes, note thecorrect barcodes used, and consider reanalyzing the multiplexed samples
with the correct Bio Sample names for the barcodes actually used. If you
reanalyze the data, ensure that the Barcode Name File includes only thecorrect barcodes used.
• Mean Read Length Distribution: Histogram distribution of the mean
polymerase read length for all samples.
– Good performance: The distribution should be normal with a relatively
tight range.
– Questionable performance: A spread out distribution, with a mode
towards the low end.

Barcodes > Barcode Quality Scores• Barcode Quality Score Distribution: Histogram distribution of barcode
Quality scores. The scores range from 0-100, with 100 being a perfect match.
Any significant modes or accumulation of scores <40 suggests issues withsome of the barcode analyses. The red line is set at 26 – the minimum default
barcode score.
– Good performance: Distributions with a mode >65 and the low-end tailtapering off below 40.

Page 12

– Questionable performance: A bimodal distribution with a large second
peak usually indicates that some barcodes that were sequenced were notincluded in the barcode scoring set.

Barcodes > Barcoded Read Binned Histograms• Read Length Distribution By Barcode: Histogram distribution of thePolymerase read length by barcode. Each column of rectangles is similar to aread length histogram rotated vertically, seen from the top. Each sample
should have similar Polymerase read length distribution. Non-smoothchanges in the pattern looking from left to right might indicate suboptimal
performance.
• Barcode Quality Distribution By Barcode: Histogram distribution of the perbarcode version of the Read Length Distribution by Barcode histogram.
The histogram should contain a single cluster of hot spots in each column. Allbarcodes should also have similar profiles; significant differences in thepattern moving from left to right might indicate suboptimal performance.
– Good performance: All columns show a single cluster of hot spots.
– Questionable performance: A bimodal distribution would indicate missing
barcodes in the scoring set.

Data > File Downloads
The following files are available on the Analysis Results page. Additional
files are available on the SMRT Link server, in the analysis output
directory.
• Analysis Log: Log information for the analysis execution.
• SMRT Link Log: Server-level analysis log information. (This file is displayedwhen you choose Data > SMRT Link Log.)• Barcode Files: Barcoded subread Data Sets; one file per barcode.
• Barcode Summary CSV: Data displayed in the reports, in CSV format. This
includes Bio Sample Name.
• Barcode Summary: Text file listing how many ZMWs were filtered, how many
ZMWs are the same or different, and how many reads were filtered.
• Inferred Barcodes: Inferred barcodes used in the analysis. The barcoding
algorithm looks at the first 35,000 ZMWs, then selects barcodes with ≥10
counts and mean scores ≥45.
• Unbarcoded Reads: BAM file containing reads not associated with abarcode.

Note: You can get the demultiplexed BAM files using the DataManagement module’s “Export Data Sets” feature. In the demultiplexed

Page 13

BAM output file a tag bc is added for each read, indicating the assigned
barcode. The bc tag is the zero-based index of the barcodes in the FASTA
file. For example, when using the barcodes RSII_96_barcodes, asubread with barcode lbc1 identified on both sides will have the tag
bc:B:S,0,0 in the BAM output file.
A second bq tag corresponds to the barcode quality (0-100).
For details on running the Demultiplex Barcodes application using thecommand-line, see the document SMRT Tools Reference Guide (v9.0),
available here.

Working with
Demultiplexing
Results

Finding Demultiplexed subreads.bam Files
Subreads.bam files cannot be downloaded from the SMRT Analysis
Report page. You can find these files using the command line bynavigating to the SMRT Link server. To do so:
1. Copy the job path (Analysis Overview > Status > Path) on the SMRT

Link server shown on the Report page.

2. Open the command line, login into the SMRT Link Server and change

directory to the path you copied:$ cd /pbi/analysis/smrtlink/release/smrtsuite/userdata/jobs_root/0000/0000029/
0000029399

3. Display the folders and files in the directory of the Barcode Demultiplexing Run:$ ls -llrwxrwxrwx 1 fas Domain Users
140 Oct 10 09:36 cromwell-job -> /pbi/analysis/smrtlink/release/smrtsuite/userdata/jobs_root.local/cromwell-executions/pb_demux_subreads/
c9cf0a1c-5fef-46d9-97e4-3ad44d80b597
drwxr-xr-x 2 fas Domain Users 4096 Oct 10 09:36 entry-points
drwxr-xr-x 3 fas Domain Users 4096 Oct 10 10:04 logs
drwxr-xr-x 2 fas Domain Users 20480 Oct 10 10:04 outputs-rw-r--r-- 1 fas Domain Users
0 Oct 10 09:36 pbscala-job.stderr-rw-r--r-- 1 fas Domain Users 22778 Oct 10 10:05 pbscala-job.stdout
drwxr-xr-x 2 fas Domain Users 4096 Oct 10 10:04 workflow

Page 14

Notice that the cromwell job is a hyperlink to another directory, designed
to save space on your SMRT Link server.
4. Navigate to the subdirectory where the .bam files are stored:$cd cromwell-job/call-demultiplex_barcodes/demultiplex_barcodes/083b56b3-dee2-44aeaa97-e6656bb4763f/call-lima/execution$ls -l-rw-r--r-- 1 fas Domain Users 215561027 Oct 10 09:58 lima.bc1001--bc1001.bam-rw-r--r-- 1 fas Domain Users
388923 Oct 10 09:58 lima.bc1001--bc1001.bam.pbi-rw-r--r-- 1 fas Domain Users
12857 Oct 10 09:58 lima.bc1001--bc1001.subreadset.xml-rw-r--r-- 1 fas Domain Users 227808959 Oct 10 09:58 lima.bc1002--bc1002.bam-rw-r--r-- 1 fas Domain Users
408837 Oct 10 09:58 lima.bc1002--bc1002.bam.pbi-rw-r--r-- 1 fas Domain Users
12857 Oct 10 09:58 lima.bc1002--bc1002.subreadset.xml-rw-r--r-- 1 fas Domain Users 217156999 Oct 10 09:58 lima.bc1003--bc1003.bam-rw-r--r-- 1 fas Domain Users
390196 Oct 10 09:58 lima.bc1003--bc1003.bam.pbi-rw-r--r-- 1 fas Domain Users
12857 Oct 10 09:58 lima.bc1003--bc1003.subreadset.xml-rw-r--r-- 1 fas Domain Users 238209137 Oct 10 09:58 lima.bc1004--bc1004.bam-rw-r--r-- 1 fas Domain Users
428762 Oct 10 09:58 lima.bc1004--bc1004.bam.pbi-rw-r--r-- 1 fas Domain Users
43595 Oct 10 09:58 lima.json-rw-r--r-- 1 fas Domain Users
2757 Oct 10 09:58 lima.lima.counts-rw-r--r-- 1 fas Domain Users
2977 Oct 10 09:41 lima.lima.guess-rw-r--r-- 1 fas Domain Users 433394971 Oct 10 09:58 lima.lima.report-rw-r--r-- 1 fas Domain Users
791 Oct 10 09:58 lima.lima.summary-rw-r--r-- 1 fas Domain Users 992123522 Oct 10 09:58 lima.removed.bam-rw-r--r-- 1 fas Domain Users
2413108 Oct 10 09:58 lima.removed.bam.pbi-rw-r--r-- 1 fas Domain Users
569 Oct 10 09:58 lima.removed.json-rw-r--r-- 1 fas Domain Users
12542 Oct 10 09:58 lima.removed.subreadset.xml-rw-r--r-- 1 fas Domain Users
2 Oct 10 09:58 rc-rw-r--r-- 1 fas Domain Users
4459 Oct 10 09:37 script-rw-r--r-- 1 fas Domain Users
1162 Oct 10 09:37 script.submit-rw-r--r-- 1 fas Domain Users
0 Oct 10 09:37 stderr-rw-r--r-- 1 fas Domain Users
0 Oct 10 09:37 stderr.submit-rw-r--r-- 1 fas Domain Users
0 Oct 10 09:37 stdout-rw-r--r-- 1 fas Domain Users
63 Oct 10 09:37 stdout.submit

For each demultiplexed barcode, the Barcode Demultiplexing workflow
generates three files: subread.bam, subread.bam.pbi andsubreadset.xml.
Other files are generated and display in the advanced report:lima.lima.counts, lima.lima.guess, lima.lima.report,lima.lima.summary,lima.removed.bam

The json files (lima.json and lima.remove.json) are useful for runninga user-defined workflow.
Note: For information on working with barcoded data in pbcromwell, seepage 62 of SMRT Tools Reference Guide (v9.0).

Page 15

R plotting scripts are available on github:https://github.com/pacificbiosciences/barcoding/#quality-control.
For information on working with barcoded data in lima, see:https://github.com/PacificBiosciences/barcoding.
Additional Information:• Demultiplex Barcodes Algorithm:
https://github.com/PacificBiosciences/barcoding• PacBio BAM Format Specifications:
http://pacbiofileformats.readthedocs.io/en/9.0/BAM.html

For Research Use Only. Not for use in diagnostic procedures. © Copyright 2017 - 2020, Pacific Biosciences ofCalifornia, Inc. All rights reserved. Pacific Biosciences does not sell a kit for carrying out the overall No-Amp Targeted
Sequencing method. Use of this method may require rights to third-party owned intellectual property. Information in thisdocument is subject to change without notice. Pacific Biosciences assumes no responsibility for any errors oromissions in this document. Certain notices, terms, conditions and/or use restrictions may pertain to your use of Pacific
Biosciences products and/or third party products. Please refer to the applicable Pacific Biosciences Terms andConditions of Sale and to the applicable license terms at http://www.pacificbiosciences.com/licenses.html.
Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT, SMRTbell, Iso-Seq, and Sequel are trademarks ofPacific Biosciences. BluePippin and SageELF are trademarks of Sage Science, Inc. NGS-go and NGSengine aretrademarks of GenDx. FEMTO Pulse and Fragment Analyzer are trademarks of Agilent Technologies. All othertrademarks are the sole property of their respective owners.
See https://github.com/broadinstitute/cromwell/blob/develop/LICENSE.txt for Cromwell redistribution information.
P/N 101-923-200 Version 01 (June 2020)

Page 16