Sentieon Quick Start

Welcome to Sentieon® Software Quick Start Guide. This guide is meant to help you get started quickly. You will get information for setting up the environment and running your first Sentieon® job from BWA to Variant Calling.

Download Quick Start PDF:

What do you need to get started?

To get started using Sentieon® software, you will need the following:

  1. Hardware requirements: A Linux server with the following configuration:

  • Linux running one of the following distributions or higher: RedHat/CentOS 6.5, Debian 7.7, OpenSUSE-13.2, or Ubuntu-14.04.

  • 16GB of memory for small panel or whole exome or 64GB for whole genome.

  • (Recommended) High-speed SSD drives are preferred for ideal I/O performance to get maximum CPU utilization.

  1. Software requirements:

  • Python 2.6.x, Python 2.7.x, or python3.x is required. You can check Python version by typing the following:

python --version

  1. Sentieon® software release package:

  • Download the package from the link provided by the technical support at Sentieon.

  • Decompress the package by running the following command, where VERSION is the version you are using, for example 202503.02:

    tar xvzf sentieon-genomics-VERSION.tar.gz

  1. License requirements: Please see the Appendix for more details on how to set up your license. IT support may be needed.

  2. Environment requirements:

  • If Python 2.6.x, Python 2.7.x, or python3.x is not the default Python version, you can set the following environment variable.

export SENTIEON_PYTHON=Python_location

  • If you are using a localhost license file, set the following environment variable, where LICENSE_DIR is where the license file is located, and LICENSE_FILE.lic is the license file name.

    export SENTIEON_LICENSE=LICENSE_DIR/LICENSE_FILE.lic

  • If user is using a license server, set the following environmental variable, where LICSRVR_HOST and LICSRVR_PORT are the hostname and port of the license server. Please see the next section for more details.

    export SENTIEON_LICENSE=LICSRVR_HOST:LICSRVR_PORT

  • For convenience, set the binary path as shown below, where PATH_TO_ SENTIEON_BINARY_DIRECTORY is where Sentieon® binary is installed.

    export SENTIEON_INSTALL_DIR=PATH_TO_SENTIEON_BINARY_DIRECTORY

  • For improved performance when using NFS storage, set the SENTIEON_TMPDIR environmental variable to point to local scratch fast storage.

    export SENTIEON_TMPDIR=/tmp

Start your first Sentieon® DNAscope job

Sentieon® Inc. provides a quick start package that includes a sample script and data to help you quickly test the installation and to diagnose potential problems.

The quick start package includes data for a single chromosome, both sequence data of a sample and reference materials. The job script uses the Sentieon DNAscope pipeline for a set of pair-ended Illumina fastq files:

  • BWA: Map reads to the reference.

  • Metrics and LocusCollector: Collect reads’ statistics.

  • Dedup: Remove duplicate reads.

  • Variant calling: DNAscope variant calling.

Note

DNAscope is only recommended for use with samples from diploid organisms. For other samples, please use DNAseq.

Run the quick start package

To get started, copy the downloaded quick start package to a new directory, and unpack it by running the following:

tar xzvf sentieon_quickstart.tar.gz

Here is what is included in the package:

  • sentieon_quickstart.sh: the sample shell script that drives the entire pipeline.

  • reference: a directory that contains human genome reference files and database files of known SNP sites.

  • models: a directory that contains DNAscope model files.

  • FASTQ files: sample sequence files.

Before running the script, you need to make sure that the environment variables are properly set as described above, including the license and path to the directory.

Then open your favorite editor to edit the user settings in sentieon_quickstart.sh.

# Update with the location of the Sentieon software package
SENTIEON_INSTALL_DIR=/home/release/sentieon-genomics-202503.02

# Update with the location of temporary fast storage and uncomment
#SENTIEON_TMPDIR=/tmp

# It is important to assign meaningful names in actual cases.
# It is particularly important to assign different read group names.
sample="sample_name"
group="read_group_name"
platform="ILLUMINA"

# Other settings
nt=16 #number of threads to use in computation

# Is the data prepared with a PCR free library prep
PCRFREE=true

Note

In the user setting shell script sentieon_quickstart.sh:

  • It is important to assign meaningful names in actual cases.

  • It is particularly important to assign different read group names.

To get the number of the CPU cores, user can run nproc as shown below.

nproc

To better understand the rest of the sentieon_quickstart.sh script, please read the comment in each section, and the corresponding chapters in the manual.

Now, launch the script by simply running sentieon_quickstart.sh, and watch the result unfold. The entire run takes about 3 - 5 minutes on a typical Linux server. Actual time varies depending on the computation environment.

sh sentieon_quickstart.sh &

Understanding the results

Below is a list of the files, their meaning and references. For more details, please refer to documentation.

  1. Quick start test output files

File name

Description

sorted.bam

Coordinate-sorted BAM file after alignment with Sentieon® BWA mem.

score.txt

Duplicate read data file.

aln_metrics.txt

Alignment and general statistics of the two pair sequence reads.

gc_summary.txt

GC bias statistics summary.

gc_metrics.txt, qc-report.pdf

GC bias statistics data file and report PDF.

qd_metrics.txt, qd-report.pdf

Base quality score distribution data file and report PDF.

mq_metrics.txt, mq-report.pdf

Cycle-dependence of the mean quality score data file and report PDF.

is_metrics.txt, is-report.pdf

Insert size distribution data file and report PDF.

deduped.bam

Output BAM file of Dedup stage, with duplicated reads removed.

dnascope.vcf.gz

Output VCF file of DNAscope variant calling.

Description of the Sentieon® tools

The table below shows the different Sentieon® products and tools and their purpose. It is also noted if a tool implements functionality equivalent to an existing GATK pipeline tool.

Table 1 Sentieon tools

Sentieon® product

Sentieon® tool

Typical use

Equivalent GATK pipeline tool

Sentieon® BWA

Sentieon® BWA

Read alignment and mapping

BWA

DNAscope

DNAscope

Improved germline SNV/Indel/SV calling

DNAseq®

Genotyper

Germline SNV/Indel calling, non haplotype based

UnifiedGenotyper

DNAseq®

Haplotyper

Germline SNV/Indel calling

HaplotypeCaller

DNAseq®

GVCFtyper

Joint calling of cohorts, demonstrated up to 200,000 samples

GenotypeGVCFs

DNAseq®

VarCal

Calculate Variant Quality Score Recalibration

VariantRecalibrator

DNAseq®

ApplyVarCal

Apply Variant Quality Score Recalibration

ApplyRecalibration

RNAseq

RNASplitReadsAtJunction

RNA SNV/Indel calling

SplitNCigarReads

RNAseq

Haplotyper

RNA SNV/Indel calling

HaplotypeCaller

TNseq®

TNsnv

Somatic SNV calling, non haplotype based

MuTect

TNseq®

TNhaplotyper

Somatic SNV/Indel calling

MuTect2

TNseq®

TNhaplotyper2 + TNfilter

Somatic SNV/Indel calling

Mutect2 and FilterMutectCalls from GATK4

TNscope®

TNscope®

Improved somatic SNV/Indel/SV calling

General tools

Dedup and LocusCollector

Perform deduplication

Picard MarkDuplicates

General tools

Realigner

Perform Indel relaignment for non-haplotype based callers

RealignerTargetCreator and IndelRealigner

General tools

QualCal

Perform Base Quality Score Recalibration

BaseRecalibrator, AnalyzeCovariates

General tools

ReadWriter

Create BAM files

PrintReads

General tools

AlignmentStat

QC metrics

Picard CollectAlignmentSummaryMetrics

General tools

BaseDistributionByCycle

QC metrics

Picard CollectBaseDistributionByCycle

General tools

CollectVCMetrics

QC metrics

Picard CollectVariantCallingMetrics

General tools

ContaminationAssessment

QC metrics

ContEst

General tools

CoverageMetrics

QC metrics

DepthOfCoverage

General tools

GCBias

QC metrics

Picard CollectGcBiasMetrics

General tools

HsMetricAlgo

QC metrics

Picard CollectHsMetrics

General tools

InsertSizeMetricAlgo

QC metrics

Picard CollectInsertSizeMetrics

General tools

MeanQualityByCycle

QC metrics

Picard MeanQualityByCycle

General tools

QualDistribution

QC metrics

Picard QualityScoreDistribution

General tools

QualityYield

QC metrics

Picard CollectQualityYieldMetrics

General tools

SequenceArtifactMetricsAlgo

QC metrics

Picard CollectSequencingArtifactMetrics, ConvertSequencingArtifactToOxoG

General tools

WgsMetricsAlgo

QC metrics

Picard CollectWgsMetrics

Appendix - Set up license

Sentieon® software is a license-controlled software. The user is required to properly set up the license in order to run the software.

We provide two types of the licenses:

  • Single machine evaluation license: this license is used for evaluating the Sentieon® software in a single machine. It allows new users to get quickly started on using the software without requiring help from the IT department. In order to use this license, the computer where you plan on running the Sentieon® software requires external Internet access.

  • Cluster license: this license is used in a cluster environment. With this license, a floating license server lightweight process is running on one node in the cluster, serving licenses though TCP to all other nodes that have network connection to the license server. This license server is running in a special non-computing node on the cluster periphery that has unrestricted access to the outside world through HTTPS, and serves the licenses to the rest of the nodes in the cluster by listening to a specific TCP port that needs to be open within the cluster.

Setting up a single machine evaluation license

To use the single machine evaluation license, the computing node needs have access to the Internet. This allows Sentieon® software to validate the license.

To use a single machine evaluation license, follow the steps below:

  1. Copy the license file to the computing node. For example, the license file LICENSE_FILE.lic is now located at LICENSE_DIR .

  2. Set up environment variable as below:

export SENTIEON_LICENSE=LICENSE_DIR/LICENSE_FILE.lic

Setting up license server

As shown in Fig. 1, license server requires the following:

  1. The license server should have access to the Internet to perform license validation.

  2. The computing nodes should have access to the license server via a host name LICSRVR_HOST

  3. The machine the license server is running has an open port for the license services to listen on, and the computing nodes have access to that port. Here we assume the available port is LICSRVR_PORT

../_images/quick_start-fig1.png

Fig. 1 Topology of the computing nodes and license server

You may need IT support to get LICSRVR_HOST:LICSRVR_PORT, and confirm that the above requirements are met.

Note

If the license server is behind a firewall, separated from the computing nodes through a NAT, the license server’s hostname/IP visible to the nodes may be different from its actual hostname/IP. If this is the case, you will need to bind the license server on the actual IP address, while the compute node requests license from the IP address after NAT. Please contact Sentieon support for more details.

Follow these steps to obtain license file, set up and test the license server:

  1. Send the following information to Sentieon® to receive the license file:

  • FQDN (fully qualified domain name) LICSRVR_HOST of the designated machine to run license service.

  • The designated port LICSRVR_PORT to Sentieon® to receive the license file.

  1. Copy the received license file to the license server LICSRVR_HOST. We assume the license file is located in LICENSE_PATH/LICENSE_FILE. Run the following command on the license server to start the license server process:

    <SENTIEON_INSTALL_DIR>/bin/sentieon licsrvr --start --log LOG_FILE LICENSE_PATH/LICENSE_FILE

  2. Alternatively, you can follow the instructions in section 8.8 - Running the license server (LICSRVR) as a system service in the Sentieon® Genomics Manual, to configure and start the license server as a system daemon.

  3. Go to the Sentieon® installation directory. Run the following commands on the license server to confirm the license server is up and running.

    <SENTIEON_INSTALL_DIR>/bin/sentieon licclnt ping -s LICSRVR_HOST:LICSRVR_PORT

    If the command returns without an error message, the license server is up and running.

  4. Login to one of the computing node, go to the Sentieon® installation directory, and run the above command again:

    <SENTIEON_INSTALL_DIR>/bin/sentieon licclnt ping -s LICSRVR_HOST:LICSRVR_PORT

    If the command returns without an error message, the computing node now can access the license server, too.

  5. Set up the following environment variable and you are good to go.

    export SENTIEON_LICENSE=LICSRVR_HOST:LICSRVR_PORT