Illumina Sequencing: How To Plan Your First Sequencing Project

Illumina® Sequencing:
How to Plan Your First

Sequencing Project
Illumina Technical Support

Objectives
• We will discuss the comprehensive workflow for planning your first sequencing project
• Upfront considerations
• Library preparation
• Sequencing
• Analysis
• This is an overview of many topics, so I will include helpful resources along the way for
more in depth information
2
Upfront Considerations
Overview of the sequencing process
Nucleic Library Data

Sequencing
Acid Prep Analysis
4
Nucleic acid
• The nucleic acid of interest will affect all of the subsequent steps
• We will first need to know the source, some examples are:
• Genomic DNA
• Cell-free DNA
cell
• Total RNA
• Metagenomic samples
• FFPE DNA or RNA
• ChIP DNA
• Many more…
5
Nucleic acid
• We also need to ensure high purity of the sample

• It is important to know how much nucleic acid we will have, and if it is degraded, as
these factors can influence our next steps
6
Resources
• Introduction to Illumina® NGS

• www.illumina.com/science/technology/next-generation-sequencing.html
• DNA/RNA isolation considerations for Illumina® Library Preparation kits
• support.illumina.com/bulletins/2016/05/dnarna-isolation-considerations-when-using-truseq-
library-prep-kits.html
• DNA sequencing
• www.illumina.com/techniques/sequencing/dna-sequencing.html
• RNA sequencing
• www.illumina.com/techniques/sequencing/rna-sequencing.html
• FFPE sequencing
• www.illumina.com/science/education/ffpe-sample-analysis.html
7
Library Preparation
Library preparation - overview
• Library Preparation methods

• How to choose the right library prep method for your project
• The steps of library preparation
• Library quantification and QC
• Library preparation resources
• Library pooling
9
Library preparation methods
• Whole genome libraries

• de novo or resequencing
• RNA-seq libraries
• mRNA-seq, total RNA-seq, small RNA-seq
• Shotgun metagenomics
• Sequencing multiple genomes or transcriptomes from the same sample
• Bisulfite libraries
• Discover sites of DNA methylation
• Customized libraries
10
For Research Use Only. Not for use in diagnostic procedures.
Library preparation methods – continued
• Amplicon libraries
• targeted library preparation, for when we are interested in specific genes or variants
• Enriched libraries
• larger targeted panels, many genes or full exomes
11
Library Prep and Array Kit Selector
www.illumina.com/library-prep-array-kit-selector.html
For Research Use Only. Not for use in diagnostic procedures. 12

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14
15
16

Resources
• Library Preparation and Array kit selector

• www.illumina.com/library-prep-array-kit-selector.html
• Sequencing Method Explorer
• www.illumina.com/science/sequencing-method-explorer.html
• Create custom targeted sequencing panels with DesignStudio
• designstudio.illumina.com/
• Illumina® Sales;
• illuminabrasil@illumina.com
• +55 113500.3900 option 2
18
Library Preparation
For clustering: For sequencing: For pooling samples:

Libraries must have P5 and Libraries must have Libraries must have a
P7 binding regions on sequencing primer unique index or barcode
either end of a library binding regions sequence
19
Library flow cell binding and priming
https://support.illumina.com/downloads/indexed-sequencing-overview-15057455.html?langsel=/us/
20
Library quantification and QC
• Quantification
• Accurate library quantification is critical
• Will affect the amount of data going to all samples
• Is a key factor in sequencing run performance in terms of data output and data quality
• Quantification methods
• Each workflow/protocol will have a recommended quantification method, such as qPCR or
fluorometric quantification
• We do not recommend using UV spectrophotometers
21
Library quantification and QC
• Quality Control
• Instruments like the BioAnalyzer and Fragment Analyzer allow us to visualize the size
distribution of libraries in displays called traces
• We can run traces to assess library success and determine average fragment length
• We can use gels for some library types
22
Library preparation support
https://support.illumina.com/sequencing/sequencing_kits/illumina-dna-prep.html

Library pooling
• Library pooling can also be referred to as multiplexing

• We typically pool multiple libraries together for a single sequencing run
• Sequencing runs produce enough sequencing reads for multiple samples, for most
applications
• The number of libraries to pool will depend on the amount of data we need for each
library.
• The fewer libraries pooled together for sequencing, the greater the data each library will get
24
Library pooling
• How do I determine how many libraries to pool?

• Sequencing Coverage Calculator:
• support.illumina.com/downloads/sequencing_coverage_calculator.html
• Library Preparation and Array kit selector:
• www.illumina.com/library-prep-array-kit-selector.html
25
Sequencing Coverage Calculator
support.illumina.com/downloads/sequencing_coverage_calculator.html
26







Library normalization and pooling
• We want to bring all libraries to the same concentration (in nanomolar) before pooling;
this is normalization
• Normalization is how we ensure even data for all libraries within the same sequencing
run
• Some workflows include normalization within the protocol (integrated); for others, we
must manually normalize the samples
34
Library normalization and pooling
• Once all samples are normalized, we can pool the samples together
• Pool equal volumes of each library
• We recommend pooling at least 5 ul of each
• Pipetting ≥5 ul improves pooling accuracy
• May result in a larger pool than is required for sequencing
35
Resources
• Library quantification and quality control quick reference guide support bulletin
• support.illumina.com/bulletins/2016/05/library-quantification-and-quality-control-quick-
reference-guide.html
• Best practices for manually normalizing library concentrations support bulletin
• support.illumina.com/bulletins/2017/03/best-practices-for-manually-normalizing-library-
concentrations.html
• Recommended library normalization method for the iSeqTM 100 system support
bulletin
• support.illumina.com/bulletins/2019/07/recommended-library-normalization-method-for-the-
iseq-100-system.html
• Illumina® Pooling Calculator
• support.illumina.com/help/pooling-calculator/pooling-calculator.htm
36
Resources
• Sample multiplexing overview

• www.illumina.com/science/technology/next-generation-sequencing/multiplex-
sequencing.html
• Estimating Sequencing Coverage
• www.illumina.com/documents/products/technotes/technote_coverage_calculation.pdf
• Optimizing Coverage for Targeted Resequencing
• support.illumina.com/content/dam/illumina-
marketing/documents/products/technotes/technote_optimizing_coverage_for_targeted
_resequencing.pdf
• Index Adapters Pooling Guide
• support.illumina.com/downloads/index-adapters-pooling-guide-1000000041074.html
Sequencing
Sequencing - overview
• Where to sequence
• How to choose the appropriate sequencer for you project
• How to set up a sequencing run
• Resources for sequencing and instruments
39
Where to sequence?
• You may have the sequencer you would like to use in your lab
• You may want to use an outside sequencing service or core facility
• Academic and other facilities
• Some facilities will do library preparation and sequencing
• Each core will have submission requirements
40
Choosing the right sequencer
• How much data is needed?
• How many libraries will I sequence?
41
Illumina sequencing platforms
Focused Power Flexible Power
iSeqTM 100 MiniSeqTM MiSeqTM NextSeqTM 550 NextSeqTM 1000/2000
Population Power
NovaSeqTM 6000 NovaSeqTM X NovaSeqTM X Plus

Choosing the right sequencer
www.illumina.com/systems/sequencing-platforms/comparison-tool.html
43
How to set up a sequencing run
• Plan run configuration

• Prepare sequencing consumables
• Denature and dilute library pool
• Load libraries and consumables onto the sequencer
44
Plan run configuration
• We will decide on the sequencing read lengths

• For example: 2x150 bp
• We will list library and indexing information
• This will identify each sample by the index sequence we added during library preparation
• We may specify some analysis settings
Illumina® Local BaseSpace

BaseSpace
Experiment Run Instrument
Prep Tab
Manager Manager Run Setup
45
Plan run configuration
• Illumina® Experiment Manager

• support.illumina.com/sequencing/sequencing_software/experiment_manager.html
• Local Run Manager
• support.illumina.com/sequencing/sequencing_software/local-run-manager.html
• BaseSpaceTM PrepTab
• basespace.illumina.com/lab
46
Prepare sequencing consumables
• Thaw sequencing reagents

• At room temperature in a water bath
• At room temperature in air
• At 4ºC
• Bring flow cell to room temperature
47
Denature and dilute library pool
• Libraries must be single stranded for sequencing

• Each instrument has a protocol for denaturing and diluting library pool before loading
• Some instruments will denature the libraries for you, for others, you will need to denature the
samples prior to starting the run
• Dilute libraries to loading concentration
• Each instrument has a range of loading concentrations
• Add PhiX control library to library pool
• PhiX spike-in is used as a sequencing control
• Place pool on ice until loading the sequencer
48
Load libraries and consumables
• For most sequencers, we will add libraries to the reagent cartridge in a dedicated position
• The instrument control software will walk us through loading the flow cell, reagent
cartridge, buffer, and emptying the waste container
• Once the sequencer is loaded, pre-run checks will start
• After the pre-run checks pass, we start the sequencing run
49
Resources
• Illumina® Sequencing by Synthesis (SBS) technology

• www.illumina.com/science/technology/next-generation-sequencing/sequencing-
technology.html
• SBS video
• www.illumina.com/company/video-hub/fCd6B5HRaZ8.html
• Sequencing workflows on different platforms
• support.illumina.com/downloads/indexed-sequencing-overview-15057455.html
• More information on thawing a storing sequencing reagents
• support.illumina.com/bulletins/2016/07/how-to-thaw-and-store-sequencing-reagents-for-
optimal-performance-.html
50
Reviewing run performance
• BaseSpaceTM Sequence Hub

• If the sequencing run has been sent to BaseSpace, or if we are using BaseSpace run
monitoring
• https://basespace.illumina.com/
• Sequence Analysis Viewer
• Sequence Analysis Viewer uses files from the sequencing run folder to visualize run
performance
• support.illumina.com/sequencing/sequencing_software/sequencing_analysis_viewer_sav.html
• What do we want to check?
• Data quality
• Data output
51
Instrument resources





support.illumina.com/sequencing/sequencing_instruments/miseq.html

Data Analysis
Data analysis - overview
• FASTQ generation and demultiplexing

• Next steps in data analysis
• Software options for data analysis
• Data analysis resources
59
FASTQ generation and demultiplexing
• FASTQ generation
• Convert raw sequencing output into As, Ts, Cs, and Gs with associated quality scores
• Demultiplexing
• Separate data for each sample by index
60
FASTQ generation and demultiplexing
• FASTQ generation and demultiplexing will proceed automatically for:

• MiSeq™ runs
• iSeq™ 100 runs
• MiniSeq™ runs
• NextSeq™ 500/550 runs set up with Local Run Manager
• Any runs set up to upload to BaseSpace™ Sequence Hub during run set up
• NextSeq™ 550 and NovaSeq™ 6000 runs not set up to stream to BaseSpace™
Sequence Hub will need to be demultiplexed separately
• bcl2fastq software
• NextSeq™ 1000/2000
• Includes on-instrument and BaseSpace™ Sequence Hub options
61
Next steps in data analysis
• De novo assembly
• Alignment to a reference sequence
• Variant calling
• Counting reads
62
Options for data analysis – Local Run Manager
Generate DNA RNA DNA

FASTQ Amplicon Amplicon Enrichment
TruSightTM Amplicon Resequencing Small RNA

Tumor 15 DS Analysis RNA Fusion
PCR 16S Assembly Library

Amplicon Metagenomics Analysis QC

Options for data analysis – BaseSpaceTM Sequence Hub

DRAGEN Analysis Apps

Resources
• Introduction to Key Concepts in Sequencing Data Analysis Webinar

• https://support.illumina.com/training.html
• BaseSpaceTM Sequence Hub public data sets
• basespace.illumina.com/datacentral
66
Resources
• BaseSpaceTM Sequence Hub

• basespace.illumina.com
• Local Run Manager
• support.illumina.com/sequencing/sequencing_software/local-run-manager.html
• MiSeqTM Reporter
• support.illumina.com/sequencing/sequencing_software/miseq_reporter.html
• bcl2fastq
• support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html
67
Summary
• Upfront sequencing consideration

• Library preparation
• Sequencing
• Data analysis
68
Additional resources
• Additional Technical Support Webinars can be found on the Support Webinar page here:
• https://support.illumina.com/training.html
• Bulletins can be found here:
• support.illumina.com/bulletins.html
• Publication reviews:
• www.illumina.com/science/publication-reviews.html
69
Additional resources
• A beginner’s guide to NGS

• www.illumina.com/science/technology/next-generation-sequencing/beginners.html
• Contact Illumina® Tech Support for help:

• techsupport@illumina.com;
• +55 (11) 3500.3939 – (Monday – Friday 8am to 5pm).
70
For additional assistance please contact us at the link below:
https://www.illumina.com/company/contact-us.html#
© 2022 Illumina, Inc. All rights reserved.

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Illumina® Sequencing:

How to Plan Your First

Illumina Technical Support

Nucleic Library Data

• We also need to ensure high purity of the sample

• Introduction to Illumina® NGS

• Library Preparation methods

• Whole genome libraries

For Research Use Only. Not for use in diagnostic procedures. 12

For Research Use Only. Not for use in diagnostic procedures. 17

• Library Preparation and Array kit selector

For clustering: For sequencing: For pooling samples:

• We can use gels for some library types

For Research Use Only. Not for use in diagnostic procedures. 23

• Library pooling can also be referred to as multiplexing

• How do I determine how many libraries to pool?

For Research Use Only. Not for use in diagnostic procedures. 27

For Research Use Only. Not for use in diagnostic procedures. 28

For Research Use Only. Not for use in diagnostic procedures. 29

For Research Use Only. Not for use in diagnostic procedures. 30

For Research Use Only. Not for use in diagnostic procedures. 31

For Research Use Only. Not for use in diagnostic procedures. 32

For Research Use Only. Not for use in diagnostic procedures. 33

• Sample multiplexing overview

• How much data is needed?

• How many libraries will I sequence?

iSeqTM 100 MiniSeqTM MiSeqTM NextSeqTM 550 NextSeqTM 1000/2000

NovaSeqTM 6000 NovaSeqTM X NovaSeqTM X Plus

• Plan run configuration

• We will decide on the sequencing read lengths

Illumina® Local BaseSpace

• Illumina® Experiment Manager

• Thaw sequencing reagents

• Libraries must be single stranded for sequencing

• Illumina® Sequencing by Synthesis (SBS) technology

• BaseSpaceTM Sequence Hub

For Research Use Only. Not for use in diagnostic procedures. 52

For Research Use Only. Not for use in diagnostic procedures. 53

For Research Use Only. Not for use in diagnostic procedures. 54

For Research Use Only. Not for use in diagnostic procedures. 55

For Research Use Only. Not for use in diagnostic procedures. 56

For Research Use Only. Not for use in diagnostic procedures. 57

• FASTQ generation and demultiplexing

• FASTQ generation and demultiplexing will proceed automatically for:

Generate DNA RNA DNA

TruSightTM Amplicon Resequencing Small RNA

PCR 16S Assembly Library

For Research Use Only. Not for use in diagnostic procedures. 63

For Research Use Only. Not for use in diagnostic procedures. 64

For Research Use Only. Not for use in diagnostic procedures. 65

• Introduction to Key Concepts in Sequencing Data Analysis Webinar

• BaseSpaceTM Sequence Hub

• Upfront sequencing consideration

• A beginner’s guide to NGS

• Contact Illumina® Tech Support for help:

© 2022 Illumina, Inc. All rights reserved.

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