Roche Oncology Analysis Server User guide

Brand: Roche

Model: Oncology Analysis Server

Type: User guide

Roche Oncology Analysis Server This high-performance next-generation sequencing data analysis server streamlines your analysis workflow by enabling you to run multiple pipelines in parallel while maintaining high data quality through a comprehensive set of quality control metrics. The intuitive user interface provides you with full control over your analysis parameters and allows you to easily review and manage your results. The scalable architecture ensures that the server can grow with your increasing throughput needs.

AVENIO Surveillance &

Monitoring Software

User Guide

Version 1.0

Software Version 1.0

For Research Use Only. Not for use in diagnostic procedures.

Publication information

AVENIO Surveillance & Monitoring Software User Guide, v1.0

Publication information

Publication version

Software version

Revision date

Change description

1.0

June 2020

First version

Edition notice

This publication is intended for users of the AVENIO Surveillance & Monitoring Software.

Every effort has been made to ensure that all the information contained in this publication is correct at the time of publishing. However, the

manufacturer of this product may need to update the publication information as output of product surveillance activities, leading to a new

version of this publication.

Where to find information

This guide contains information about using the monitoring software, organized according to the normal operation of the program.

The AVENIO Oncology Analysis Software User Guide focuses on routine operation of the AVENIO Oncology Analysis Software. This document

can be downloaded from PIM.web.

The white paper titled In-depth assessment reveals powerful performance and flexibility of the AVENIO ctDNA Analysis Kits (file name

SEQ100108_AVENIO ctDNA_Performance_White_Paper) includes information on performance. This document can be downloaded from

https://sequencing.roche.com/content/dam/rochesequence/worldwide/resources/SEQ100108_AVENIO%20ctDNA_Performance_White_Paper.pdf

Screenshots

The screenshots in this publication have been added exclusively for illustration purposes. Configurable and variable data, such as tests, results,

or path names visible therein must not be used for laboratory purposes.

Trademarks

The following trademarks are acknowledged:

AVENIO is a trademark of Roche.

All other trademarks are the property of their respective owners.

Contact addresses

Manufacturer

Roche Molecular Systems, Inc.

1080 US Highway 202 South

Branchburg, NJ 08876

USA

Distribution

Roche Diagnostics GmbH

Sandhofer Strasse 116

68305 Mannheim

Germany

Distribution in USA

Roche Diagnostics

9115 Hague Road

Indianapolis, Indiana 46256

USA

Technical support

For product-related technical documents or to contact Roche Technical Support, go to sequencing.roche.com/support.

Publication information

AVENIO Surveillance & Monitoring Software User Guide, v1.0

Table of contents

Publication information ....................................................................................................................................... 2

Contact addresses ....................................................................................................................................................................................................... 2

Technical support ........................................................................................................................................................................................................ 2

Preface .................................................................................................................................................................... 4

Regulatory disclaimer ................................................................................................................................................................................................. 4

AVENIO Surveillance & Monitoring Software ................................................................................................................................................... 4

Symbols, abbreviations, and acronyms ............................................................................................................................................................... 4

Symbols used in this publication ..................................................................................................................................................................... 4

Abbreviations and acronyms ............................................................................................................................................................................. 4

What is new in publication version 1.0 ................................................................................................................................................................ 5

Chapter 1. Getting started .................................................................................................................................. 6

Glossary ........................................................................................................................................................................................................................... 6

Text conventions .......................................................................................................................................................................................................... 6

Chapter 2. Panel design ...................................................................................................................................... 7

Surveillance panel design ......................................................................................................................................................................................... 7

Surveillance panel background .............................................................................................................................................................................. 8

Chapter 3. Overview and background.............................................................................................................. 9

Workflow and bioinformatics pipeline overview .............................................................................................................................................. 9

Background information ........................................................................................................................................................................................... 9

Chapter 4. Installation and setup .................................................................................................................... 10

Installation requirements ........................................................................................................................................................................................ 10

Network overview and NFSv3 share setup ...................................................................................................................................................... 11

Installing the monitoring software ...................................................................................................................................................................... 11

To install the monitoring software ................................................................................................................................................................. 12

Checking the connection to the NFS server ................................................................................................................................................... 12

To check NFSv3 share mounting ................................................................................................................................................................... 12

Chapter 5. Using the monitoring software .................................................................................................... 14

Input files ...................................................................................................................................................................................................................... 14

Creating a Sample Manifest ............................................................................................................................................................................ 14

Running the monitoring software ........................................................................................................................................................................ 16

Output files ................................................................................................................................................................................................................... 17

Chapter 6. Troubleshooting .............................................................................................................................. 20

Executing the aveniosam software ..................................................................................................................................................................... 21

Interpreting unexpected results ........................................................................................................................................................................... 22

Preface

AVENIO Surveillance & Monitoring Software User Guide, v1.0

Preface

Regulatory disclaimer

For Research Use Only. Not for use in diagnostic procedures.

AVENIO Surveillance & Monitoring Software

The goal of the AVENIO Surveillance & Monitoring (SAM) Software is to enable the detection of molecular Minimal

Residual Disease (MRD) of solid tumors, based on circulating tumor DNA (ctDNA) in plasma, using tumor-specific

biomarkers (reporters). The tool leverages utilization of multiple variants (SNV and Indel) enabling detection of traces

of ctDNA in plasma derived from subjects affected by solid tumors.

AVENIO SAM allows researchers to detect minute traces of ctDNA in plasma samples after curative intent treatment

and in advanced disease in surveillance and monitoring settings respectively.

This software is a command line tool that runs on a Linux system and is recommended to be executed by a

Bioinformatician. The workstation must have access to the directory that stores the output from the AVENIO Oncology

Analysis Server runs.

The monitoring software requires a reporters list, which must be generated by the user, and the OAS output from

running one or more samples through the AVENIO Oncology Analysis Software v1.1.0 or v2.0.0. It will produce a series

of text files that provide a summary of the tumor burden and MRD estimation in each sample analyzed. These text files

are easily imported into Excel or another application for subsequent analysis.

Symbols, abbreviations, and acronyms

Except where the context clearly indicates otherwise, the following product names and descriptors are used.

Product name

Descriptor

AVENIO Surveillance & Monitoring Software

Monitoring software, SAM software

Symbols used in this publication

Symbol

Explanation

Important note: Highlights information that is critical for optimal performance of the system. May also indicate that loss of

data or invalid data could occur if the precautions or instructions are not observed.

Information note: Identifies items of general interest and additional information about the topic or procedure being

described.

Abbreviations and acronyms

The following abbreviations and acronyms are used.

Abbreviation/acronym

Definition

CSV

Comma-separated Value

ctDNA

circulating tumor DNA

EULA

End User License Agreement

FFPET

Formalin-Fixed Paraffin-Embedded Tissue

HPC

High Performance Computing

LOD

Limit of Detection

OAS

Oncology Analysis Server

MRD

Minimal Residual Disease

NFS

Network File System

RSR

Roche Service representative

SNV

Single Nucleotide Variant

VCF

Variant Call Format

Preface

AVENIO Surveillance & Monitoring Software User Guide, v1.0

What is new in publication version 1.0



First version

Chapter 1. Getting started

AVENIO Surveillance & Monitoring Software User Guide, v1.0

Chapter 1. Getting started

This guide provides common procedures and recommendations for installing and using the AVENIO Surveillance &

Monitoring Software. It is intended for customer IT/system administrators who are responsible for installing the

monitoring software on a Linux-based machine, and trained bioinformatics experts who are responsible for using and

interpreting the software.

This guide is organized by sections to help you understand, install, use, and analyze the monitoring software in

conjunction with the output provided by both the AVENIO Oncology Analysis Software v1.1.0 and v2.0.0.

The process for installing and using the software includes 4 main components:



Setup and prerequisite check



Installing the program



Running the program



Analyzing the output

Glossary

Term

Definition

Allele Fraction or Allele Frequency (AF)

The AF for a variant is defined as the ratio of the number of unique molecules that support

the variant to the total unique molecule depth at that position. It is also a quantifier of how

much a particular SNV (or Indel) variant is present. A closely related concept is “Depth” or

“Alternative Depth.”

ctDNA

Circulating tumor DNA.

Freq file

A file produced by the AVENIO Oncology Analysis Software that contains unique molecule

depths per position and DNA base type.

Indel

Insertion or deletion.

Minimal Residual Disease (MRD)

Refers to the minimal presence of ctDNA in peripheral circulation during and/or after

treatment in the absence of signs and symptoms.

MMPM

Mutant Molecules Per Milliliter.

Monitoring

Refers to serial assessment of molecular MRD in advanced metastatic cancer setting

during the course of disease.

Reporter

Tumor-specific biomarkers.

Reporter VCF File

A tumor-specific biomarker (SNV or Indel) that the user provides as input to the monitoring

software in a manner that conforms to the VCF format.

SNV

Single Nucleotide Variant.

Surveillance

Refers to serial assessment of molecular MRD after curative intent treatment during the

surveillance time period.

Tumor Burden

Refers to the amount of cancer present in the body. Quantification of ctDNA-specific

mutations in peripheral circulation has been shown to correlate with tumor burden in solid

tumors. Normalized measurements of average AF and MMPM can be utilized to reflect

tumor burden.

Text conventions

Conventions

Description

Numbered listing

Indicates steps in a procedure that must be performed in the order listed.

Courier New font

Indicates user input at the command line.

Italic type

Indicates names of files.

Italic type, within <>

Identifies variables or words that stand for values that you fill in.

Bold type, Courier New font,

within shaded gray code block

Highlights user entry within a code block that is described in the step.

Bold type, blue

Identifies names of menus and controls (buttons, checkboxes, etc.) in the software.

Italic type, blue

Indicates a hypertext link to another location within the document, or to a website.

Chapter 2. Panel design

AVENIO Surveillance & Monitoring Software User Guide, v1.0

Chapter 2. Panel design

Surveillance panel design

The surveillance panel is the ideal partner for monitoring because the panel design was optimized for monitoring.

Exons from COSMIC and other databases covering recurrent mutations in potential driver genes were selected.

Subsequently, population-scale data from multiple cancer databases was applied to provide broad mutation coverage.

The resulting panel has, on average (for lung and colorectal cancers), 4-6 mutations per patient, which provides a

4-6-fold improvement for Limit of Detection (LOD) compared to a single mutation. Newman

et al., demonstrated that

using multiple mutations enables a low ctDNA detection limit (≤0.01%) for monitoring applications.

Figure 1 shows the percentage of subjects having mutations greater than or equal to a particular mutation count.

The surveillance panel is uniquely optimized for surveillance and monitoring as it maximizes the number of mutations

detected per subject.

Figure 1 Average mutations per subject by panel

https://www.ncbi.nlm.nih.gov/pubmed/24705333

Chapter 2. Panel design

AVENIO Surveillance & Monitoring Software User Guide, v1.0

This is important because increasing the number of reporters used gives greater sensitivity for ctDNA detection as

shown in Figure 2.

Figure 2 Greater sensitivity with increased number of reporters

Surveillance panel background

The surveillance panel was specifically designed for NSCLC and CRC; however, its utility was also tested for other

indications and found to perform well based on the median number of mutations detected per subjects as shown in

Figure 3.

Figure 3 Disease area coverage by surveillance panel

Chapter 3. Overview and background

AVENIO Surveillance & Monitoring Software User Guide, v1.0

Chapter 3. Overview and background

Workflow and bioinformatics pipeline overview

The monitoring workflow includes the 3 steps detailed in Figure 4.

Figure 4 Monitoring software workflow

1. Generation of Reporter Files (timepoint 0) – Reporter files represent the tumor-specific biomarkers (reporters)

that the user is interested in monitoring. This file is a VCF file generated by the customer derived from tumor

profiling using a baseline sample (for example, Time point 0). This can be generated using the AVENIO ctDNA

or Tumor Tissue product or curated from customer data. Reporter lists generated from customer data must be

intersected with the surveillance or expanded panel to generate results for AVENIO SAM. We recommend the

use of matched normal samples to remove germline and CHIP variants to ensure a tumor specific reporter list.

2. Processing of TimePoint Samples – Plasma samples collected at subsequent timepoints (for example,

Timepoint 1) must be processed through the AVENIO software. The SNV and Indel output files produced by

the AVENIO software will be used as input to the monitoring software.

3. Execution of Monitoring Software – The reporter files and sample input files are run through the monitoring

software, which generates monitoring results for SNVs, Indels, and Synthesis (overall results merging SNV and

Indel results). The results files are used for surveillance and monitoring in these samples.

Background information

The following information is available from your Roche affiliate upon request.



AACR 2018 Poster presents a use case for monitoring.



AMP 2017 Poster about the AVENIO ctDNA kit highlights the use of the kit for monitoring.



CRC Surveillance 2017 Poster details an example use case for the surveillance panel.

Chapter 4. Installation and setup

AVENIO Surveillance & Monitoring Software User Guide, v1.0

Chapter 4. Installation and setup

Installation requirements

The following requirements are necessary for installing the monitoring software:



Linux-based machine

• 16 GB of free memory (32 GB installed)



An installed and running version of Docker 1.12 or higher. In early 2017, Docker changed to a new versioning

scheme, stating that Docker version 17.03 will supersede version 1.13. Therefore, any Docker version installed

after the versioning scheme change (17.03 or higher) will also meet the necessary requirements for the

AVENIO Surveillance & Monitoring Software.

• To check if Docker is already installed on the remote machine, use the following command:

$ docker run hello-world

If Docker is installed, the message Hello from Docker! appears.

If Docker is not installed, install Docker by following the instructions below for your Linux version.

o https://docs.docker.com/install/linux/docker-ce/centos/

o https://docs.docker.com/install/linux/docker-ce/debian/

o https://docs.docker.com/install/linux/docker-ce/fedora/

o https://docs.docker.com/install/linux/docker-ce/ubuntu/

To test the successful installation of Docker, repeat the command above:

$ docker run hello-world

If Docker was installed successfully, the message Hello from Docker! appears. If the installation was not

successful, contact the professional who manages Docker at your local site or your system administrator.



A terminal emulator (for example, PuTTY) that allows for an SSH connection from your computer to the remote

machine hosting the monitoring software.



The internal directory of the OAS output. The monitoring software needs the output from the OAS to retain the

same directory structure to process the data.

Chapter 4. Installation and setup

AVENIO Surveillance & Monitoring Software User Guide, v1.0

Network overview and NFSv3 share setup

Figure 5 shows the working process for installation, configuration, and utilization of the AVENIO Surveillance and

Monitoring Software (including integration with the AVENIO Oncology Analysis Software).

Figure 5 Working process

1. Customer gets the software from the RSR – The software will be installed at the customer site by an RSR.

This will be either via a download or USB.

2. Customer installs the monitoring software – The RSR will install the software on the customer workstation or

lab network.

3. Customer configures the monitoring run – Following the instructions in Chapter 5, the customer will

prepare the input files for the monitoring software run. The monitoring software requires read access to the

AVENIO Oncology Analysis Software output files.

4. User launches the monitoring run – A user with bioinformatics expertise will execute the monitoring run.

5. Monitoring run produces output reports – The monitoring software will generate a set of output files that

show the tumor burden based on the reporters found in the sample. These reporters are written to the directory

from which the monitoring software was run.

Installing the monitoring software



The Docker image (aveniosam.image.tar) downloaded from Google Drive is not redistributable by any customers to

any other parties.



The Docker image (aveniosam.image.tar) downloaded from Google Drive is provided as-is and must not be subject

to any kind of modification by the user side before or after it is installed on their system. In particular, we advise

that the installation be performed by a credited system administrator who has the admin privilege to install a

Docker image to that particular system.

Chapter 4. Installation and setup

AVENIO Surveillance & Monitoring Software User Guide, v1.0

To install the monitoring software

1. After downloading the software from the Google Drive folder, navigate to the directory where the image.tar and

bin.zip files reside.

2. Decompress the bin.zip file, using the following command:

$ unzip bin.zip

3. Copy the aveniosam executable from the appropriate subdirectory into the same directory where the image.tar

file is located. The following is an example of copying the file for 64-bit systems.

$ cp ./bin/linux-amd64/aveniosam .

4. Install the software using the following command:

$ aveniosam install

$ ./aveniosam install

5. Read and agree to the End User License Agreement, and wait for the installation to finish (5-10 minutes).

At this point in the installation, the Roche Service representative (RSR) must provide

the

End User License Agreement (EULA) for the customer to read, review, and agree to.

The customer should type yes to acknowledge reading the EULA before the RSR

initiates the final step of the installation.

Checking the connection to the NFS server

The monitoring software requires network read access from the output location configured for

the

OAS. It is imperative that the NFSv3 share be mounted on the same machine running the

monitoring software to allow it to function correctly. The following step

s provide guidance to

confirm connectivity, configure and mount the share locally, and perform a test run to ensure

the software is functioning

correctly.

Use ping to confirm connectivity from the machine running the monitoring software to the NFS server containing the

output from the OAS. This action can be completed using either the NFS server’s hostname (if available) or its statically

assigned IP address (example shown below):

$ ping hostname

$ ping 192.168.1.1

If neither option works, verify that the NFS server is working and has the same IP address that was used in the original

installation setup of the OAS.

To check NFSv3 share mounting

1. Mount the NFSv3 share on the same machine that the monitoring software is installed. A typical example of this

command is as follows:

$ sudo mount.nfs -o vers=3 <hostname:/nfs export path> /tmp/<temp mount directory>

This command mounts the NFS content on the local machine.

Chapter 4. Installation and setup

AVENIO Surveillance & Monitoring Software User Guide, v1.0

2. Using the cd and ls commands, verify that you can navigate to and browse the content that is shared by the

NFSv3 server.

$ cd/tmp/<temp mount directory>

$ ls -l

3. Confirm that the machine has access to the OAS output directory mounted on the NFS server. The output of the

ls command ran previously in step 2 is used to confirm accessibility to the OAS output directory. If the directory

listing is given, then access has been achieved. If a permissions error occurs, or the directory contents are unable

to be listed using the ls command, then additional configuration and troubleshooting may be required.

Chapter 5. Using the monitoring software

AVENIO Surveillance & Monitoring Software User Guide, v1.0

Chapter 5. Using the monitoring software

The monitoring software is a command line tool that can be installed locally on a Linux workstation or on a computer

cluster for wider access. It requires a manifest file, reporters file, and OAS outputs for samples processed on AVENIO

expanded or surveillance panel.

Input files

Creating a Sample Manifest

The extractedMass_ng, inputVol_ml, and medianDedupDepth should be obtained from the pipeline run associated with

the sample being monitored.

• extractedMass_ng – taken from the Isolated DNA Mass (ng) field in the manifest file used for the analysis run

• inputVol_ml – taken from the Plasma Volume (mL) field in the manifest file used for the analysis run

• medianDedupDepth – taken from the Unique Depth Median field in the SampleMetrics.csv download

Ensure that you use the same medianDedupDepth in the monitoring sample manifest as the value generated from the

sample pipeline run. Using a different value will result in an increase or decrease of the Mutant Molecule value in the

SNV, Indel, and Synthesis files.

1. Sample manifest file <sample_manifest.csv>:

a. Provides information about the individual samples that will be included in the analysis.

b. A CSV (comma-separated values) formatted file that contains the following headers:

subjectID

An identifier string composed of letters, numbers, and special characters defined by the user.

The subjectID is also part of the output folder name, in the form results_subjectID. The subjectID

does not need to match those used in the AVENIO OAS Software.

Example: subject123

sampleID

An identifier string composed of letters, numbers and underscores defined by the user.

The sampleID must match the sampleID used in the AVENIO software.

Example: Spike1_30ng_1_40M

drawDay

A numerical value defined by the user representing the day within the subject observation period

that the sample was acquired.

Example: 30

sampleDir

The output directory generated by the OAS software (v1.1 or v2.0).

extractedMass_ng

Numerical value indicating the amount of sample mass isolated in nanograms.

Example: 30

inputVol_ml

Numerical value indicating the volume of sample input in milliliters.

Example: 4

medianDedupDepth

Numerical value giving the median de-duplicated read depth of the sample.

Example: 3000

2. Reporter file <reporters.yaml>:

a. Mapping file with the location of the reporter list for SNV and Indel.

b. File is in yaml format. YAML is a human-readable data-serialization language. It is commonly used for

configuration files and in applications where data is being stored or transmitted.

Chapter 5. Using the monitoring software

AVENIO Surveillance & Monitoring Software User Guide, v1.0

c. File contains the following reporter lists:

o snvReportersVcf (SNV reporter list is the SNV reporters to be monitored in VCF format)

o indelReportersVcf (Indel reporter list is the Indel reporters to be monitoring in VCF format)

snvReportersVcf: "/tmp/test/monitoring/reporters/snv.reporter.vcf"

indelReportersVcf: "/tmp/test/monitoring/reporters/indel.vcf"

where the text before the ":" is the name of the reporter list being referenced, and the text inside the quotation

marks is the absolute file path to the location of the reporter file.

The VCF formatted files listed in the Reporter file contain the following headers:

CHROM

The name of the sequence (typically a chromosome) on which the variation is being called.

Example: chr4

POS

The 1-based position of the variation on the given sequence.

Example: 56315463

The identifier of the variation.

Example: dbSNP

REF

Reference allele.

Example: C

ALT

Alternate or mutant allele.

Example: T

QUAL

A quality score associated with the inference of the given alleles.

Example: 100

FILTER

A flag indicating which of a given set of filters the variation has passed.

Example: PASS

INFO

An extensible list of key-value pairs (fields) describing the variation.

Example: DP=2049; ALTDP=924

FORMAT

An (optional) extensible list of fields for describing the samples.

SAMPLE

For each (optional) sample described in the file, values are given for the fields listed in FORMAT

d. Can selectively monitor a subset of variants (e.g., only SNV itself or SNV and Indel):

o To monitor SNV, provide snvReporters.vcf

o To monitor Indel, provide indelReporters.vcf

o To monitor both SNVs and Indels, specify both reporter files in the yaml file

3. Samples are plasma samples collected at specific time-points and run through the AVENIO Surveillance or

Expanded Kit.

Chapter 5. Using the monitoring software

AVENIO Surveillance & Monitoring Software User Guide, v1.0

Running the monitoring software



Example syntax without passing the first argument flag will result in the following help menu of possible first

argument values.

$ aveniosam –m sample_manifest.csv -p surveillance -r reporters.yaml

where:

Usage:

Aveniosam [command]

Available commands:

exec

Execute the workflow

help

Help about any command

install

Install the longitudinal analysis Docker image

license

View End User License Agreement

uninstall

Uninstall the image

version

View version info

Flags:

-h, --help

Help for Aveniosam



Example syntax to run the monitoring software is as follows:

$ aveniosam exec -m sample_manifest.csv -p surveillance -r reporters.yaml

where:

Usage:

Aveniosam exec [flags]

is the executable program that is called by the user

Aliases:

Exec, workflow

is an argument passed to the executable instructing it to run the monitoring

software

Flags:

-h, --help

is help for exec

-m, --manifest-file string

is manifest file (required)

-p, --panel string

is panel name (required), acceptable values (all lowercase): {expanded,

surveillance}

-r, --reporters-file string

is reporters file (required)

All 3 flags (-m, -p, -r) and corresponding arguments or files are required. No other flags are allowed. If any of

the required flags or arguments/files are missing, the program will not run and exit with an error message.



The sample manifest file must contain the following columns in a CSV format (as specified under Input files). This

directory is named in the format results_subjectID.

• subjectID

• sampleID

• drawDay

• sampleDir

• extractedMass_ng (this can be found in the manifest file used for the analysis run)

• inputVol_ml (this can be found in the manifest file used for the analysis run)

• medianDedupDepth (this can be found in the SampleMetrics.csv download)



The output from the monitoring software will be placed in the same directory from where the command is initiated.

Chapter 5. Using the monitoring software

AVENIO Surveillance & Monitoring Software User Guide, v1.0

Output files

The results are available in a newly created directory where the command was run. This directory is named results.

Inside the results_subjectID directory, there should be four files with different names all with the .txt extension type.

The details of these files can be found below:



monitor_synthesis.txt



monitor_snvs_varpos.txt



monitor_snvs_summary.txt



monitor_indels.txt

The top-level directory contains the 4 files that summarize the monitoring results:



monitor_synthesis.txt is a subject-level, tab-delimited file containing 1 row entry per monitored sample that

summarizes the detected variant allele fraction, mutant molecules per milliliter, p-value, and other monitoring

outputs for each sample. This file contains the overall results, which combine the output from the SNV and Indel

components. OVERALL_PVAL is calculated by combining the p-value for SNV and p-value for Indels using

Fisher’s method.

Field Name

Field Description

SUBJECT

Subject

SAMPLE

Sample

OVERALL_AF Combined

Average Allele Fraction of all SNV and Indel reporters

MAX_AF

Maximum Allele Fraction

OVERALL_MMPM

Mean Mutant Molecules per mL for all variants

SUM_MMPM

Sum of Indel and SNV MMPM values

OVERALL_TMPM

(dedupDepth * extractedMass_ng * 330) / (medianDedupDepth *

inputVol_ml)

dedupDepth is the alt depth of the variant

MAX_TMPM

(dedupDepth * extractedMass_ng * 330) / (medianDedupDepth *

inputVol_ml)

dedupDepth is the total depth of the mutant molecules

NORM_MMPM

SUM_MMPM/SUM_TMPM

OVERALL_PVAL

Overall P-Value (the number used to define the cutoff at which to

accept the values reported for OVERALL_AF and OVERALL_MMPM)

SNV_REPORTER_COUNT

Number of SNV Reporters

SNV_NONZERO_COUNT

Number of SNV Reporters Found

SNV_AF

SNV Mean Allele Fraction

SNV_MMPM

Overall P-Value (the number used to define the cutoff at which to

accept the values reported for SNV_AF and SNV_MMPM)

SNV_PVAL

SNV P-Value

INDEL_REPORTER_COUNT

Number of Indel Reporters

INDEL_NONZERO_COUNT

Number of Indel Reporters Found

INDEL_AF

Indel Mean Allele Fraction

INDEL_MMPM

Overall P-Value (the number used to define the cutoff at which to

accept the values reported for INDEL_AF and INDEL_MMPM)

INDEL_PVAL

Indel P-Value

Chapter 5. Using the monitoring software

AVENIO Surveillance & Monitoring Software User Guide, v1.0



monitor_snvs_summary.txt is a subject-level, tab-delimited file containing 1 row entry per monitored sample that

summarizes the detected variant allele fraction, mutant molecules per milliliter, p-value, and other SNV monitoring

outputs for each sample.

Field Name

Field Description

SUBJECT

Subject

SAMPLE

Sample

AF_MEAN

Mean Allele Fraction

SUM_MM

Total/Sum Mutant Molecules

NZ_COUNT

SNV Non-Zero Count

MM_PVAL

P-Value based on Mutant Molecules

AF_PVAL

P-Value based on Allele Fraction

NZ_PVAL

P-Value based on Non-Zero Count

SIM_AF_MEAN

Used for troubleshooting by internal support

SIM_SUM_MM_MEAN

Used for troubleshooting by internal support

SIM_NZ_MEAN

Used for troubleshooting by internal support

SIM_AF_MEAN_95PCT

Used for troubleshooting by internal support

SIM_SUM_MM_95PCT

Used for troubleshooting by internal support

SIM_NZ_95PCT

Used for troubleshooting by internal support

NUM_REPS

Number of reporters



monitor_snvs_varpos.txt is a variant-level tab-delimited file containing 1 row entry per tumor-specific SNV per

monitored sample that shows the variant allele fraction, mutant molecules per milliliter, variant allele deduplicated

sequencing depth, and total deduplicated sequencing depth at that variant position.

Field Name

Field Description

SUBJECT

Subject

SAMPLE

Sample

CHR

Chromosome

POS

Position on Chromosome

REF

Reference Allele

ALT

Alternative Allele

DEPTH

Depth at Reference Allele

VARDEPTH

Variant Depth (Alternative Allele)

Alternative Allele Fraction

MMPM

Mutant Molecules per mL

TMPM

(dedupDepth * extractedMass_ng * 330) / (medianDedupDepth * inputVol_ml)

dedupDepth is the total depth

REF_PLUS

Reference Plus Strand

REF_MINUS

Reference Minus Strand

ALT_PLUS

Alternative Allele Plus Strand

ALT_MINUS

Alternative Allele Minus Strand

Chapter 5. Using the monitoring software

AVENIO Surveillance & Monitoring Software User Guide, v1.0



monitor_indels.txt is a variant-level tab-delimited file containing 1 row entry per tumor-specific indel per

monitored sample that shows the variant allele fraction, mutant molecules per milliliter, variant allele deduplicated

sequencing depth, total deduplicated sequencing depth at that variant position, and Z score (statistical significance).

ZSCORE is calculated by combining the plus_zscore and minus_zscore using Stouffer’s method.

Field Name

Field Description

SUBJECT

Subject

SAMPLE

Sample

CHR

Chromosome

POS

Position on Chromosome

REF

Reference Allele

ALT

Alternative Allele

DEPTH

Depth at Reference Allele

VARDEPTH

Variant Depth (Alternative Allele)

Alternative Allele Fraction

MMPM

Mutant Molecules per mL

TMPM

Total Mutant Molecules per mL

ZSCORE

Combined Z-score on both strands

PLUS_ZSCORE

Z-score on the plus strand

MINUS_ZSCORE

Z-score on the minus strand

In that same directory, there is a folder named with the Subject ID, which contains the intermediate files used to

generate the summary files. These can be used if you are interested in the raw output from the SNV, Indel, or

Synthesis modules used in the monitoring software.

Output directory structure and Results files

The monitoring software produces output files that summarize the results. The output directory is named

results_[subject]. The output directory structure is as follows:

results_[subject]/

├── monitor_indels.txt

├── monitor_snvs_summary.txt

├── monitor_snvs_varpos.txt

├── monitor_synthesis.txt

└── Subject-1

├── monitor_synthesis.json

├── Spike1_30ng_1

│ ├── fusion_out.json

│ ├── fusion_out_withsubject.txt

│ ├── indel_out.json

│ ├── indel_out_withsubject.txt

│ ├── snv_out.json

│ ├── snv_out_varpos_withsubject.txt

│ └── snv_out_withsubject.txt

└── Spike2_30ng_1

├── fusion_out.json

├── fusion_out_withsubject.txt

├── indel_out.json

├── indel_out_withsubject.txt

├── snv_out.json

├── snv_out_varpos_withsubject.txt

└── snv_out_withsubject.txt

Chapter 6. Troubleshooting

AVENIO Surveillance & Monitoring Software User Guide, v1.0

Chapter 6. Troubleshooting

You cannot run multiple instances of aveniosam on the same machine; otherwise, the following error

message

will be given.

docker: Error response from daemon: Conflict. The name

"/aveniosamwf_0.6.0" is already in use by container

1ac801e664f5f33b23e49c992293ae142764460ae9d5be49d333aeba6cce1f5

c. You

have to remove (or rename) that container to be able to reuse that name..

See 'docker run

--help'.

2019/07/15 10:22:02 docker: Error response from daemon: Conflict. The

name "/aveniosamwf_0.6.0" is already in use by container

1ac801e664f5f33b23e49c9

92293ae142764460ae9d5be49d333aeba6cce1f5c. You

have to remove (or rename) that container to be able to reuse that name..

See 'docker run

--help'.

2019/07/15 10:22:02 Error running the workflow

2019/07/15 10:22:02 exit status 125

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