Hello my name is Ivan Godinez, Senior
Technical Application Scientist at Illumina,
and today I’m going to walk us through the
TruSeq Custom Amplicon solution.
Which is a
streamlined workflow to perform targeted
next-generation sequencing studies.
I’m going to walk you through the entire
process, starting with the designing of your
panel, prepping your libraries for
sequencing, and running the MiniSeq System,
and analyzing your data.
So let’s get started.
The first step is designing your custom
panel using DesignStudio.
This process is
quite similar to what you’re used to doing
with Sanger sequencing.
DesignStudio is a free and easy-to-use tool
that allows you to create custom
enrichment, targeted RNA, and custom
DNA amplicon designs.
It’s fast and
provides high target coverage for even
tough to design regions.
The program is simply accessed
through the Illumina website.
You can design up to 1536 targets, but if
you’re interested in designing more targets
than that, you can contact our Illumina
Concierge Services group.
There’s a lot of
flexibility in amplicon size.
If you have questions or issues
anywhere along the way,
experts are always available to help.
You can play around and create as many
designs as you want.
Start from your myillumina account.
Go to illumina.com, and if you don’t have an
account, create one.
It’s easy to start a design.
The toolbar
across the top helps guide you through the
process with intuitive, visible buttons.
First choose the type of sequencing.
In this example, we’ll choose amplicon.
Next, go to start your design.
In this case,
we’re selecting DNA.
Choose your assay type,
which will be TruSeq Custom Amplicon.
There’s also a convenient tool
to help you choose your assay type.
Then, select the species you’re working with.
In this case, we’re choosing human,
but you have many different options.
If you are working with non-model
organisms and yours is not listed here,
contact our Concierge team.
They will be able to work with your organism of interest.
Next, name your design.
If you'd like to, you can add a description.
We’ll select
MiniSeq for the instrument and low input
for the assay version.
Because we’ll be
sequencing on a MiniSeq System for this
project with low-input samples, we’ll
choose these two options.
If you are working with low input and/or
FFPE-derived DNA, TruSeq Custom
Amplicon Low Input is right for you.
All other applications you can choose low
input or version 1.5.
The buttons are easy to see, and there are
help buttons along the way if you ever get
stuck or need assistance.
You'll find this to be quite similar to designing targets for
your qPCR and Sanger sequencing assays,
so the process is really not too different
from what you already know.
Next, you’ll add your targets.
You can do this 4 different ways.
The tabs under “add targets” include 4 options.
First, add your gene name.
It automatically populates.
It’s very simple.
Or, you can add free text by listing your
targets or coordinates by gene or
coordinate, click “add targets,” then it will
appear on the screen.
The third option to
add targets is by doing coordinates.
The fourth and final option is adding a file.
You can do so by adding a file that contains
gene names and/or coordinates.
Save either as a CSV or TSV file, then browse and upload it.
The first and the final options are very
similar to what you’re used to doing with
other technologies.
Next, click submit.
It will tell you the
estimated number of amplicons, base
pairs, and a summary of everything you’ve
chosen up to this point.
The software is now processing your design.
It should take less than 60 minutes
depending on how many targets you have.
You’ll receive an email when your design is
ready, so you can step away and do
something else in the meantime.
When your design is ready, you can review it.
The most useful metric for assessing
your design is coverage.
You should be aiming for 95% or greater coverage.
If lower, you can contact our Concierge
Services group for further assistance in
designing your assay.
You can also see your regions of interest
that were not covered, if anything was missed.
You can visualize your gaps using
the UCSC genome browser.
Again, if you
ever need help, you can go right to the
bottom bar on the page.
Next, click “order.”
If you’re happy with
your design, the order button takes you
right to your Illumina account.
Here you can choose how many samples you want to order.
From there, you get the pricing details.
Either request a quote or add to your cart.
For library prep, the first step is hybridization of the probes you just designed.
The second step, extension/ligation, starts
at probe 1 and adds bases in.
A DNA
Polymerase extends bases all the way to
the downstream probe, then a ligase
comes in and finishes the job by ligating
the extended piece to the downstream
probe.
Then the ligase comes in and fills the gap.
Now you have your amplicon.
The third step is PCR where you add your
indexes and adapter.
The final step is to
amplify it.
There are multiple ways to set up and
analyze your run.
The MiniSeq includes
Local Run Manager for streamlined on-
instrument analysis.
However, today, we’ll
use BaseSpace Sequence Hub.
BaseSpace Sequence Hub allows you to set
up a run by entering the library prep
method and sequencing run parameters.
Once the run is created, you can find it in
the Select Run section of the MiniSeq
workflow.
As the data is generated, it will
automatically upload to BaseSpace
Sequence Hub.
Next, we’ll load our prepared library and
reagents into the MiniSeq System.
The MiniSeq is our smallest, simplest, and
most affordable sequencing platform.
The MiniSeq is highly flexible, supporting a
wide range of DNA and RNA applications
with both predefined and custom panels.
Examine multiple targets or entire
pathways in a single run, eliminating the
iterative testing of Sanger sequencing and
qPCR.
The MiniSeq automatically performs clonal
amplification, sequencing and analysis.
You won’t need any other ancillary
equipment like an emulsion PCR system or
an analysis server.
The MiniSeq uses a single integrated
reagent cartridge that contains all the
reagents necessary for clonal
amplification, sequencing, and a post-run
wash.
All you have to do is thaw this
cartridge and load your library.
Before you start sequencing, you’ll thaw
your reagent cartridge in a room
temperature water bath for 1 hour.
Take your library and load it into the well-
marked “load library here.”
Once you’re loaded, push the sequence button on the sequencer.
You’ll then log in to your
BaseSpace Sequence Hub account.
If you don’t have an account, you can sign up for one at illumina.com.
Once you’re logged in, push NEXT.
The system will bring up any runs you
might have saved.
Next, load the instrument.
Open the flow cell compartment door,
open the flow cell clamp,
and load your new flow cell,
close the flow cell clamp, and close the door.
Open the reagent compartment door to
slide in your new reagent cartridge.
Hit next, then the system will load.
Make sure you want to do the run that you set up.
If everything looks good, hit NEXT.
Automatic tests make sure you have
enough storage, your network is working,
and reagents are loaded properly.
Once complete, the start icon will populate.
Push start, and you’re up and running.
Targeted NGS enables deep sequencing,
which allows greater confidence compared
to Sanger sequencing for calling variants in
a given region of interest.
It is the most cost-effective approach
compared to Sanger, qPCR, exome, or
whole-genome sequencing for the
interrogation of tens to thousands of
genes with a high number of samples.
All of this data requires an advanced
analysis platform.
With the BaseSpace
Informatics Suite, our cloud-based data
analysis solution, it’s simple and easy.
The platform is accessible via web-based
devices, including phones, laptops, or
tablets, from anywhere you can access the
network.
BaseSpace Sequence Hub provides
scalable storage for your genomic data.
Monitor your sequencing runs remotely,
and share your data easily.
Once you log in to BaseSpace and verify
your credentials, you’re good to go.
If you don’t have an account, you can sign up here.
You can see important stats like coverage,
number and quality of variant calls, and
alignment statistics.
Coverage uniformity
measures how evenly the sequence data is
represented given the target region.
This includes both bases sequenced and the
depth at which they were sequenced.
Again, if you need any help, click the “i” icon
BaseSpace Variant Interpreter Beta
integrates with BaseSpace Sequence Hub
to enable quick identification
and classification of disease-relevant
variants on a single platform.
The user friendly and flexible variant-to-
report workflow allows you to easily
interpret and select variants of interest to
include in customized reports tailored to
your needs.
Next, you’ll import the VCF file from
BaseSpace Sequence Hub.
After that, enter
case information to view the list of
variants, assign interpretations, and issue a report.
So in summary, today we’ve gone over
how to create a custom design with
DesignStudio, prep your libraries, perform
a sequencing run on the MiniSeq System,
and do data analysis with BaseSpace
Sequence Hub and Variant Interpreter
Beta.
We hope you’ve seen how simple and easy
it is to get started with targeted NGS using
the TruSeq Custom Amplicon solution.
For more information, contact your sales rep
or visit illumina.com.