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This method is appropriate for the following:
Prepared libraries
Samples with assigned indexes If your samples do not have indexes assigned and are not in libraries, use Import Biological Samples.
On the Libraries page, select Import.
[Optional] Create a CSV file as follows.
Select the spreadsheet image to download a template.
For each sample, enter the following information.
Sample ID—The unique sample ID.
Name—A descriptive name for the biological sample.
[Optional] Species—The appropriate species.
[Optional] Project—The name of the project to save samples to. Although optional at this step, a project is required later to store the data.
NucleicAcid—The nucleid acid, either RNA or DNA.
Well—The plate well.
Index1Name—The Index 1 name.
Index1Sequence—The Index 1 sequence.
Index2Name—The Index 2 name.
Index2Sequence—The Index 2 sequence.
Save the file.
Select Choose .csv File.
Browse to and select the appropriate CSV file, and then select Open. The information from the file populates the Import Sample Libraries page.
[Optional] Select additional libraries as follows.
Select Save & Continue Later.
Select the checkbox of each library you want to use.
Select Pool Libraries.
On the Pools page, drag a library from the plate to a pool. For Nextera Rapid Capture libraries, assign samples from the same enrichment to 1 pool.
In the Pool ID field, enter a unique name for the pool.
[OptionalSelect Add Pool to add a pool.
[Optional] Use 1 of the following methods to pool libraries from multiple plates:
Select the Plate ID drop-down arrow to switch between plates.
Select Save & Continue Later, select the checkbox of each pool to merge, and then select Merge Pools.
Select Plan Run.
To help manage multiple pools, the color of each sample well matches the color of the pool the sample was added to.
The MiniSeq, NextSeq, and NeoPrep systems provide an option to set up a run in BaseSpace Sequence Hub using the Prep tab.
Set Up a Custom Library Prep Kit
When you complete run setup in BaseSpace Sequence Hub, the run becomes available in the control software of the instrument. For instructions to complete the run, see the system guide for your instrument:
MiniSeq System Guide (document # 1000000002695)
NextSeq 550 System Guide (document # 15069765)
NextSeq 500 System Guide (document # 15046563)
NeoPrep System Guide (document # 15049720)
Use the following steps to import biological samples from an external .csv file.
From the runs page, select New Run, and then select Prep Tab.
From the Prep page, select Biological Samples.
Select Import.
[Optional] Create a .csv file as follows.
Select the spreadsheet image to download a template.
Complete the following fields for each sample:
Sample ID—Enter a unique sample ID.
Name—Enter a descriptive name for the biological sample.
[Optional] Species—Enter the appropriate species.
Project—Enter the name of the project to save samples to. The selected project is the default project that contains biosample data output.
Nucleic Acid—Enter RNA or DNA.
Save the file.
Select Choose .csv File.
Browse to the appropriate file and select Open. Information from the .csv file populates the Biological Samples page.
[Optional] Select additional samples as follows.
Select Save & Continue Later.
Select the checkbox for each sample you want to use.
Select Prep Libraries.
Complete the following steps to create biological samples one at a time. For information about adding multiple samples, see Import Biological Samples.
Select the Prep tab, and then select Biological Samples.
Select Create.
Provide the following information:
Sample ID—Enter a unique sample ID.
Name—Enter a descriptive name for the biological sample.
[Optional] Species—Select a species from the drop-down list.
Project—Select Select Project to select or create a project to add your samples to. The selected project is the default project that contains biosample data output.
Nucleic Acid—Select the type of nucleic acid.
[Optional] Select additional samples as follows.
Select Save & Continue Later.
Select the checkbox for each sample you want to use.
Select Prep Libraries.
BaseSpace Sequence Hub converts sample name underscores to dashes in the output FASTQ files. To avoid unexpected FASTQ file names, use only alphanumeric characters and dashes in sample names.
From the Runs page, select New Run, and then select Prep Tab.
From the Prep page, select NeoPrep.
From the NeoPrep Runs page, select Create New Run to open the Configure Run screen.
Select the Protocol drop-down arrow, and select a protocol, either TruSeq Nano DNA or TruSeq Stranded mRNA.
Select the Version drop-down arrow, and select a version of NeoPrep control software.
[Optional] In the Notes field, enter any notes.
In the Run Name field, enter a name for the run.
Select the Default Project field, select the project to configure for the run, and then select Confirm.
Select the checkbox for each process you want to run:
Prep Library—Requires preparation of libraries.
Quantify—Quantifies libraries after library prep is complete.
Normalize—Normalizes libraries after quantification is complete.
Complete the following fields as applicable. Read-only or unavailable fields are default selections for the run.
Select the Sample Count drop-down arrow, and select the number of samples to include in the run.
Select the Index Type drop-down arrow, and select the indexing scheme for the samples.
Select the Insert Size drop-down arrow, and select the insert size of the libraries. Mixed insert sizes are specified on the next page.
Select the PCR Cycles drop-down arrow, and select the number of PCR cycles for the run.
Select Next.
In the Review Run Details page, review the run parameters.
If the parameters are acceptable, select Finish.
[Optional] Select the run setup details or library card mapping to view or print details.
Select Done to return to the NeoPrep Runs page.
The new run is listed with a status of Ready, which means the run is listed in the NeoPrep control software.
When prepping a library, select + Custom Library Prep Kit in the Library Prep Kit dropdown menu. The Custom Library Prep Kit Definition page opens.
Fill out the name of the custom prep. It has the following requirements:
Unique for your account.
Characters: only alphanumeric, hyphen, underscores, and spaces accepted.
Less than or equal to 50 characters.
Select at least 1 of the supported read types.
Select at least 1 of the indexing strategies. Only selecting None is not allowed.
Fill out the default number of cycles.
Select template to download the index definition file template.
Fill out the Settings section the following way:
For single read only: no adapter (blank), or 1 adapter sequence for Read 1.
For paired-end: no adapter (blank), or 2 adapter sequences, 1 for Read 1 and 1 for Read 2.
Each adapter sequence meets the following criteria:
Sequence of A, T, C, or G character.
Length from 1 to 20 characters.
Fill out the Index1Sequences and Index2Sequences sections the following way:
For Single Index, with or without None: 1 to 100 Index 1 names
For each Index 1 name an associated Index 1 sequence
For Dual Index, with or without None and Single Index: 1 to 100 Index 1 names
For each Index 1 name an associated Index 1 sequence: 1 to 100 Index 2 names
For each Index 2 name an associated Index 2 sequence
Each index name meets the following criteria:
Unique within the file
Length from 1 to 8 characters alphanumeric, hyphen, or underscore characters.
Each index sequence meets the following criteria
Sequence of A, T, C, or G characters
Length from 1 to 20 characters
All index sequence lengths (Read 1 and Read 2) are equal
Index 1 sequences are unique within the file set of Index 1 sequences
Index 2 sequences are unique within the file set of Index 2 sequences
If the supported indexing strategy specifies Single Index, you can set up Default Layout By Well the following way:
Each well unique from A01 to H12
For each well, an associated index name exists in the specified Index1Sequences section
If the supported indexing strategy specifies Single Index or Dual Index, you can set up Default Layout By Column the following way:
Each column number unique from 1 to 12
For each column, an associated index name exists in the specified Index1Sequences section
If the supported indexing strategy specifies Dual Index, you can set up Default Layout By Row the following way:
Each row letter unique from A to H
For each row, an associated index name exists in the specified Index2Sequences section
Select the Choose.csv File button to select and upload your custom index file.
Select Create New Kit to complete the process.
Your custom library prep has been added to the library kit drop-down!
In the Assign Samples to Wells page, set up each well as follows.
In the Sample ID field, select Select Sample,
In the Select a Biological Sample dialog box, select a sample, and then select Select.
In the Library ID field, enter a unique name for the library.
Select the Index drop-down arrow, and select a unique index to add to the sample.
If you selected mixed insert sizes on the previous page, select the Insert Size drop-down arrow, and select the insert size of the library.
Select Next.
On the Plan Run page, click the Select Instrument drop-down arrow, and select a sequencing system, either MiniSeq or NextSeq.
In the Name field, enter a name for the sequencing run.
[Optional] In the Reagent Barcode field, enter the barcode ID of the reagent kit used for the run. Entering the barcode ID links the reagent kit to the run.
[Optional] Select Use Custom Primer options:
R1 — Use custom primer for Read 1.
R2 — Use custom primer for Read 2.
Select a read type, either Single Read or Paired End.
Enter the number of cycles for each read in the sequencing run:
Read 1 Cycles — Enter a value up to 151 cycles.
Read 2 Cycles — Enter a value up to 151 cycles. This value is typically the same number of cycles as Read 1.
Review the indexing scheme for the run. To make changes, override the defaults as follows.
Select the Override default indexing scheme checkbox.
Select an indexing scheme:
Single Index — Performs a run with 1 index read.
Dual Index — Performs a run with 2 index reads.
No Index — Performs a non-indexed run.
Enter the number of cycles for each index read*:
Index 1 Cycles — Enter the number of cycles required for the Index 1 (i7) primer.
Index 2 Cycles — Enter the number of cycles required for the Index 2 (i5) primer.
Make sure that a pool is present.
Select 1 of the following buttons to continue:
Sequence — The run appears on the Planned Runs list with a status of Ready. The run becomes available from the control software of your sequencing system.
Save and Continue Later — The run appears on the Planned Runs list with a status of Planning. When you are ready to sequence, select the checkbox for the run and select Sequence. The run then becomes available from the control software of your sequencing system.
*Indexing is required when sequencing multiple libraries.
If your kit is not listed but is compatible with your sequencing system, set up a custom library prep kit or select a kit that uses the same index adapter set. For example, select Nextera Rapid Capture for TruSight Cardio.
In the Libraries page, select the Library Prep Kit drop-down arrow, and select a kit.
In the Plate ID field, enter a unique plate ID.
[Optional] In the Notes field, enter any notes. For Nextera Rapid Capture libraries, specify that the plate contains multiple enrichments.
[Optional] Assign the sample to a different project.
Select the checkbox of each library to assign to a project.
Select Set Project.
In the Select Project dialog, select or create a project to assign libraries to, and then select Select.
Select Auto Prep to automatically assign each library to a well and index location. For Nextera Rapid Capture libraries, samples belonging to the same enrichment must be in the same row. Each plate has a maximum layout of 12 indexes by 8 indexes. If you require more wells or indexes, create multiple plates.
[Optional] To change indexes, select an index on the plate and select a new index from the drop-down list. NOTE: This option is not available for fixed layout plates.
[Optional] Move libraries to a different well as follows. Select the checkbox of the library to move. Drag the selected library from the Libraries area to the appropriate well.
Select Export to save your library prep settings in a .csv file. This file serves as a reference when preparing samples in the lab.
[Optional] Select additional plates as follows.
SelectSave and Continue Later to return to Libraries.
Select the checkbox of each library you want to use.
Select Pool Libraries.
Biological samples are assigned an index even if you are not performing indexed sequencing. During run setup, you can specify no indexing.
The library prep run setup for NeoPrep includes the following three parts:
Create and configure the run.
Assign samples to wells.
Review the run setup.
At any point during this process, you can select the Save button to save your work. Saved runs appear on the NeoPrep Runs page with a status of Planning. When you are ready to resume work, select the run name.