Prepare Sample Plate and Generate Manifest File

Before you begin Day 1 activities, prepare the sample plate, scan the sample barcodes, and generate a sample manifest file for the Illumina Protein Prep run.

When preparing the sample plate, work quickly to avoid thaw. Best practice is to keep the sample matrix rack and the control tubes on dry ice during the plate preparation and manifest generation steps.

Avoid processing plasma and serum samples together in the same plate. Processing plasma and serum in the same plate results in erroneous data.

The manifest file is a comma-separated values (CSV) file that provides the Illumina Protein Prep Automation System (IPPAS) with information about the input samples. Only CSV files are accepted. There are no file naming constraints.

During the automated Sample Prep procedure, you must upload the manifest file to the Illumina Lab Automation Software Solution (ILASS). Make sure to save your completed file to a location that is accessible from the IPPAS computer.

Excel regional settings determine whether a comma (,) or semicolon (;) is used as the default delimiter for separating items in a file. Before saving your manifest file, you might need to adjust the settings to make sure that a comma is used as the delimiter.

• Sample matrix rack (barcoded).
• Matrix tubes containing the samples.
• Sample manifest template file for your sample type (plasma or serum). Download the appropriate template from the Illumina support site or from the ILASS Batch Creation screen in the Sample Prep workflow.
• The following controls:
-90°C to -70°C Storage

Reagent

Box Name

Instructions

Blank

Illumina Protein Prep (IPP), Proteomics Assay, Plasma/Serum -80°C Box

While the controls are frozen, add them to the sample plate. For details, refer to Prepare the Sample Plate.

Plasma/Serum Calibrator (C-P/C-S)

Plasma/Serum QC Sample (Q-P/Q-S)

The manifest file contains the data columns listed in the following table. The columns appear beneath the header row in the file and are presented in the order specified in the table. Do not delete columns or add new columns to the manifest template file.

The manifest file columns are included in the IPPAS output file that is generated after automated library preparation is completed. ILASS adds additional information to the output file during automated library preparation.

Manifest File Column

Description

Plate Barcode

The unique barcode of the sample matrix rack. The barcode must be 8–20 characters in length. Only letters, numbers, dashes, and underscores are permitted. Every row of the manifest file must contain the same plate barcode value.

[Optional]

Matrix Tube Barcode

The unique tube barcode. The barcode must be 8–20 characters in length. Only letters, numbers, dashes, and underscores are permitted.

Well Position

The well position for the sample. All 96 positions must be present and unique. Permitted values: A01–H12

Sample ID

The sample identifier for samples and controls (Blank, Calibrator, and QC). Only letters, numbers, dashes, and underscores are permitted. The maximum number of characters permitted is 100. If you are combining two plates for sequencing, each sample ID must be unique per sample sheet.

• [Optional] You can append a globally unique identifier to control sample IDs to make sure that they are always unique. The Suffix Control Samples ID with GUID checkbox in the ILASS System Configuration screen controls this feature. When the checkbox is selected, ILASS appends identifiers automatically for all runs.

Input Type

The input sample type. Permitted values: Blank, Plasma, Plasma_Calibrator, Plasma_QC, Serum, Serum_Calibrator, Serum_QC

• The sample plate must contain at least three Blank, five Calibrator, and three QC input types.
• The input type in the first row of the manifest file must match the PLASMADIL/SERUMDIL barcode scanned during the automated Sample Prep workflow. The ILASS validates this input type match after the barcodes are scanned.
1. Create a copy of the sample manifest template file.
2. Fill out the manifest file columns for your sample plate. Follow the guidance provided in the Manifest File Contents table.
3. Save the file (CSV format).
4. Proceed as follows:
• If you intend to scan the matrix tube barcodes, save the file to the barcode scanner computer in preparation for scanning.
• If you do not intend to scan the matrix tube barcodes, save the file to a location that is accessible from the IPPAS computer.
1. Remove the controls from the box.
2. While still frozen, distribute the Blank, Calibrator, and QC controls in the sample plate as follows.
• Blank controls: B02, D06, G11
• Calibrator controls: A01, C03, G05, H08, D12
• QC controls: E04, F07, B10
• Place samples into the remaining well locations.
• For well locations that you cannot fill with a sample, place a matrix tube containing 55 µl Assay Buffer into that position. In the manifest file, refer to this input type as Blank.
 

01

02

03

04

05

06

07

08

09

10

11

12

A

Calibrator

 

 

 

 

 

 

 

 

 

 

 

B

 

Blank

 

 

 

 

 

 

 

QC

 

 

C

 

 

Calibrator

 

 

 

 

 

 

 

 

 

D

 

 

 

 

 

Blank

 

 

 

 

 

Calibrator

E

 

 

 

QC

 

 

 

 

 

 

 

 

F

 

 

 

 

 

 

QC

 

 

 

 

 

G

 

 

 

 

Calibrator

 

 

 

 

 

Blank

 

H

 

 

 

 

 

 

 

Calibrator

 

 

 

 
3. [Optional] Proceed immediately to scan the sample barcodes. Refer to [Optional] Scan the Matrix Tube Barcodes.
4. Store the sample matrix rack at ‐80°C until ready to begin Prepare for Protocol steps.

If scanning the matrix tube barcodes, proceed as follows.

1. Place the sample matrix rack on the scanner.

Make sure that the A1 tube positions for the sample matrix rack and the scanner are aligned.

2. Use the barcode scanner to scan the barcode on each sample tube.
3. Open the sample manifest template file. Copy and paste the scanned barcodes into the Matrix Tube Barcode column.

Make sure that the order of the barcodes aligns with the well positions listed in the manifest file.

4. Save the completed manifest file (CSV format) to a location that is accessible from the IPPAS computer.
5. Store the sample matrix rack at ‐80°C until ready to begin Prepare for Protocol steps.