Thermo Fisher Scientific Genexus Reference Guide

QUICK REFERENCE

Genexus™ Integr

ated Sequencer

Catalog Number A45727

Pub. No. MAN0017912 Rev. C.0

Note: For safety and biohazard guidelines, see the “Safety” appendix in the Genexus™ Integrated Sequencer User Guide (Pub. No. MAN0017910). Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

The Ion Torrent™ Genexus™ Integrated Sequencer integrates library preparation, templating, sequencing, and data analysis into a single-instrument automated run. For more information on creating assays, adding or importing samples and library batches, creating plans for sample and library runs, starting a sequencing run, and data analysis, see the Genexus™ Integrated Sequencer User Guide (Pub. No. MAN0017910). This quick reference assumes familiarity with Genexus™ Software and the Genexus Integrated Sequencer, and is intended for more experienced users.

Create an assay ................................. 1

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Add samples and create a run plan in Genexus™ Software

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Dilute the samples and load the sample plate .......... 3

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Load the sequencer and start a run .................. 4

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Review data and results ........................... 7

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Limited product warranty .......................... 7

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™

3. In the Create Sample

ﬁelds.

Attributes identiﬁed with a red asterisk (*) in the Create Sample dialog box are required when adding a new sample.

If attribute information is not available when adding a new sample, substitute mock information to complete the required ﬁelds.

4. Click Save.

The new sample is listed in the Manage Samples screen and will be available to use in your run.

dialog box, complete the required

Import samples

Sample data ﬁles can be used to capture, manage, and edit sample data. You can import sample data ﬁles in the following formats: TXT, XLS, XLSX, or CSV. For a list of the sample attributes that are included in the import ﬁle, and for information on downloading a Microsoft™ Excel™ example ﬁle to create an import ﬁle, see the Genexus™ Integrated Sequencer User Guide (Pub. No. MAN0017910), or Genexus™ Software 6.2 Help.

1. In the menu bar, click Samples4Manage Samples, then click

2. In the Import Samples ﬁle.

Import Samples.

dialog box, click Select samples

Create an assay

For information on how to create and manage assays in Genexus Software, see the Genexus™ Integrated Sequencer User Guide (Pub. No. MAN0017910). If you are using a system-installed assay without change, proceed to “Add samples and create a run plan in Genexus™ Software”.

Add samples and create a run plan in Genexus Software

You can create run plans for two types of runs: sample runs that start from nucleic acid samples, and library runs that start from manually prepared libraries. Before creating a run plan in Genexus™ Software for either a sample run or a library run, you must ﬁrst enter samples in the software to assign sample names and provide other information. Alternatively, you can import sample information from a data ﬁle.

Create a new sample

1. In the menu bar, click Samples4Manage Samples.

2. In the Manage Samples screen, click

Create Sample.

3. Navigate to the ﬁle, then click Open.

™

4. Click Upload.

A progress bar followed by an import report appears. If the import process fails, an error message indicates the reason for failure (for example, an invalid character was used).

Successfully imported samples are listed in the Samples / Manage Samples screen.

Prepare a library batch

A library batch is a group of prepared libraries that are sequenced in the same library run. If you are planning a run starting from libraries that you have already prepared manually, you must ﬁrst create a library batch in Genexus™ Software from samples that you have added. If you are planning a run starting from nucleic acid samples, skip this step and proceed to “Plan a sample run” on page 2.

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

1. In the menu bar, click Samples4Manage Samples.

2. In the Manage Samples screen, in the Filter Samples by...

dropdown menu, apply the To Be Prepared ﬁlter to limit the displayed samples to those samples that have not been placed in a library batch.

3. Select samples in the list by clicking the checkbox to the left of each sample, then click

4. In the Creat the assay that you want to run.

e Library batch screen, in Select Assay, select

Prepare Library Batch.

3. In the Assays st t

o use in the run.

Use the Filter Assays By list and the Assay Name search box to search, sort, and ﬁlter the list of assays.

4. In the Include NTC column for each assay that you select, click the Include NTC checkbox to include a no template control for the assay.

5. After you select an assay (or assays) and make the appropriate Ion Reporter™ Software selections (if applicable), click Next.

ep, select the assay or assays that you want

5. In the expanded screen, in Library Batch ID, enter a unique identiﬁer for the library batch.

6. Select the barcodes from the kit boxes into the appropriate

ﬁelds.

7. Select the Include NTC checkbox to add no template control sample processing and reporting to the library batch.

8. Type a unique library name for each DNA and/or RNA library in the appropriate ﬁeld.

9. Select the barcode ID of the adapter used to prepare each library. If appropriate, swap the default barcodes in the dialog box between DNA, RNA, and Fusions by clicking the Swap Barcodes swap image.

For example, click the DNA and Fusions swap button.

IMPORTANT! Ensure that the actual bar

used to create the libraries match the barcodes that you enter in the Create Library batch screen.

10. Enter the Input Quantity for each library.

11. Click

Submit to save and submit your selections.

The Manage Libraries screen opens, listing the library batch that you created. Libraries that are prepared in the same batch have the same Library Batch ID.

codes that you

Plan a sample run

1. In the menu bar, click Runs4Plan Sample Run.

Note: You can also click

Runs / Manage Runs screen.

2. In the

Setup step, enter or make the following selections.

a. In the Plan section, enter a unique name.

b. (Optional) In the Reporting (Optional) section, select

one or more options if needed. You can select both options, or leave both options deselected.

• Generate Report

• Upload BAM ﬁles to Ion Reporter™ Software

c. Click Next.

Plan Sample Run in the

6. In the Samples step, select the samples from the list that you want to run with the assay, then click Assign.

7. If you selected more than one assay, repeat step 6 for each additional assay.

8. If needed, edit samples in one of the following ways, then click Next.

• Click View & Remove, make your selections, then click

Update.

• Click Remove All, make your selections, then click

Assign.

9. In the Sample Plate step, review sample positions in the sample plate. Drag and drop samples and no template controls to edit the location of samples and controls, if desired.

10. Modify the concentration of samples, if needed.

11. If sample plate information is correct, click Next.

12. In the Review step, review the run plan summary, then click

Save & Print to print the run setup guide, if desired. Click Save to save the run without printing.

After saving, the run appears in the Manage Runs screen in the run list with the name you speciﬁed.

After selecting the run and loading the sequencer, the run is started on the sequencer screen.

Plan a library run

1. In the menu bar, click Runs4Plan Library Run.

Note: You can also click

Runs / Manage Runs screen.

2. In the

Setup step, enter a name for the run, then conﬁgure

the reporting options.

a. In Run Name, enter a unique name.

b. (Optional) In the Reporting (Optional) section, select

one or more options if needed. You can select both options, or leave both options deselected.

• Generate Report

• Upload Samples to Ion Reporter™ Software

c. Click Next.

Plan Library Run in the

2 Gene

egrated Sequencer Quick Reference

xus™ Int

3. In the Assays step, select the assay or assays that you want to use in the run, then click Next.

Use the Filter Assays By list and the Assay Name search box to search, sort, and ﬁlter the list of assays.

4. In the Library Batches step, select the library batch or batches that you want to use in the run.

Note: Only one library batch can be selected per assay. However, you can plan a multi-assay library run if you select multiple, dierent assays in the Assays step.

5. After you select a library batch (or batches), click Next.

6. In the Review step, review the run plan summary, then click

Save and Print to print the run setup guide, if desired. Click Save to save the run without printing.

After saving, the run appears in the run list on the Manage Runs screen with the name you speciﬁed.

The run is started on the sequencer screen after selecting a run and loading the sequencer.

Note:

If you enter a concentration <0.11 ng/µL or >10,000 ng/µL

target concentration, a warning that the concentration is out of range appears, and you are not allowed to proceed to the next step. If the concentration is ≤10,000 ng/µL, but >1,024X of the

target concentration, you can proceed, but because the instrument cannot dilute samples more than 1,024‑fold, the diluted sample concentration will be greater than the target concentration.

2. Add samples to the sample plate at the volume and positions that are speciﬁed in the run setup guide.

The sample volume is not adjustable and depends on sample type, the number of primer pools in the assay, and library chemistry. The following table also provides loading volume.

Sample type

DNA 1 15 µL

Number of primer

pools

Ion AmpliSeq™ chemistr

Volume

Dilute the samples and load the sample plate

Before starting a run on the instrument, you must quantify and dilute the samples or sample libraries, then load the sample plate.

Dilute or concentrate the samples (if needed) and load the sample plate—sample run

Isolate DNA and RNA samples using one of the procedures and kits that are recommended in the Genexus™ Integrated Sequencer User Guide (Pub. No. MAN0017910).

Samples with concentrations up to 1,024X of the target concentration for an assay (displayed as default values in the Sample Plate step screen in run planning) are in range for automated dilution and require no manual dilution. Enter the concentrations during sample run planning at the Sample Plate step (see step 9 on page 2).

1. For samples with concentrations that are out of range for automated dilution, manually dilute the sample with nuclease-free water, or concentrate the sample to a concentration ≤1,024X of the target concentration. For samples that are in range, go to step 2.

If the sample

concentr

<0.11 ng/µL Concentrate the sample to greater than or

≥0.11 ng/µL, but less than the target concentration

≤1,024X of the target concentration

ation is…

equal to the target concentration.

Run is allowed but sample concentration may not be optimal for library preparation. Concentrate the sample to greater than or equal to the target concentration.

No manual dilution is necessary. The sequencer dilutes the sample to the target concentration automatically during the run.

Then…

DNA 2 25 µL

RNA 1 15 µL

RNA 2 25 µL

Ion AmpliSeq™ HD chemistry

DNA 1 20 µL

RNA 1 20 µL

TNA 1 20 µL

3. Seal the plat

e with a sheet of Adhesive PCR Plate Foils

(Thermo Fisher Scientiﬁc Cat. No. AB0626).

4. Keep the plate on ice until ready to load it in the sequencer.

Dilute and pool libraries, and load the sample plate— library run

1. Dilute each manually prepared and quantiﬁed sample library to 200 pM with nuclease-free water.

Note: Each library must be barcoded with a unique barcode or barcode pair. Use this concentration as a starting point, then titrate up or down based on sequencing results, if needed.

2. Add equal volumes of each library to a new 1.5‑mL low DNA retention tube so that the total volume is greater than the volume speciﬁed in the run setup guide provided by the software.

Note: For information on combining DNA and RNA libraries recovered from sample runs using assays that include DNA and fusions, see the Genexus™ Integrated Sequencer User Guide (Pub. No. MAN0017910).

>1,024X of the target concentration

Genexus™ Int

Manually dilute to the target concentration based on assay type, or to a concentration in range for automated dilution by the sequencer.

egrated Sequencer Quick Reference 3

3. Mix well by pipetting up and down ﬁve times, then transfer the speciﬁed volume of each library batch to the sample plate position speciﬁed in the run setup guide.

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