For Preimplantation Genetic Screening (PGS) on Illumina ... User Manual.pdf · This kit is used to process embryo biopsy samples for preimplantation genetic screening (PGS) using

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ChromInst TM Library Preparation Kit

For Preimplantation Genetic Screening (PGS) on Illumina Platforms

Cat.#: YK016LPK-E

User Manual

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Table of Contents

Product Description……………………………………………………………………………………….…………………3

Protocol Overview…………………………………………………………………………………………….……………..3

Kit Contents……………………………………………………………………………………………………….…………….4

Equipment………………………………………………………………………………………………..………….………….4

Consumables and Equipment Supplied by the User…………………………………..…………….……….4

Important Notes………………………………………………………………………………………………………...……5

Intended Use……………………………………………………………………………………………………………… ……5

Protocol……………………………………………………………………………………………………………………….…..6

1. Cell Lysis…………………………………………………………………………………………………………………….…6

2. Pre-Library Preparation…………………………………………………………………………………………..…...7

3. Library Preparation…………………………………………………………………………………………….…………9

4. Library Pooling…………………………………………………………………………………………………………...10

5. Library Purification……………………………………………………………………………………………….…….11

6. Library Quantification……………………………………………………………………………................ .....12

7. Sequencing…………………………………………………………………………………..………………………….…12

Troubleshooting Guide……………………………………………………………………….....................…….. 13

Technical Support……………………………………………………………………………….......…………………… 14

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Product Description

This kit is used to process embryo biopsy samples for preimplantation genetic screening (PGS) using

illumina next-generation sequencing (NGS) platforms. The uniform and efficient whole-genome

amplification of single cell(s) from the biopsy sample can be achieved in a single tube with the minimal

sample loss. The streamlined and easy procedure enables NGS libraries to be prepared from biopsy

samples in about 3 hours and result reporting in approximately 9 hours*.

* Calculations based on illumina MiSeq Platform

Protocol Overview

The entire procedure includes 3 major parts: cell lysis, library preparation, and pre-sequencing sample

preparation.

Library Preparation

Cell Lysis

(14min)

Pre-Library Preparation

(70min)

Library Preparation

(41 min)

Library Pooling

(10min)

Library Purification

(45 min)

Pre-Sequencing Sample

Preparation

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Kit Contents

The kit contains enough reagents for preparing 24 library samples. Reagents are packed in 3 boxes and

should be stored at indicated temperatures upon receipt. Please make sure you receive all components

before starting the experiment.

Box 1

Note: Store all reagents at -20°C and avoid repeated freeze-thaw cycles.

Box 2

Note: Store all reagents at-20°C and avoid repeated freeze-thaw cycles.

Box 3

Note: Store reagents at 2-8°C.

Equipment

ChromInst™ Sample Prep Station (Cat.#: YK016MLB)

Consumables and Equipment Supplied by the User

Nuclease-free filter pipette tips

Nuclease-free PCR tubes, micro-centrifuge tubes

Vortexer

Benchtop microcentrifuge

Name Size & Quantity

Cell Lysis Buffer 120μl x 1

Cell Lysis Enzyme 12μl × 1

Pre-Lib Buffer 720μl × 1

Pre-Lib Enzyme Mix 25μl × 1

Library Buffer 720μl × 1

Library Enzyme Mix 20μl × 1

H2O 100μl × 1


Barcode Primer 1-24 5μl × 24


CMPure Magbeads 600μl × 1

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QubitTM Fluorometer (Thermo Fisher Scientific, Cat.#: Q33216)

Important Notes

1. Please read through the entire user manual before starting the experiment.

2. The library preparation process is very sensitive to the contamination from external DNA, so

following precautions are highly recommended for preventing or monitoring contamination during the

procedure:

Wear gloves, mask, and lab coat throughout the entire the process. Change gloves if touching one

sample before working on the next one.

Use a dedicated work area for cell lysis and preparing reaction mix of pre-library generation.

Processing the biopsy samples in a laminar flow hood is highly recommended.

Use a set of pipettes, filter tips, PCR tubes, micro-centrifuge tubes and racks, etc. only for processing

biopsy samples and preparing pre-libraries. Use another set for final library preparation, pooling and

purification.

Aliquot reagents after the first use and change tips between each liquid transfer.

Store the library products in a separate place for storing all reagents to avoid cross-contamination.

Set up a negative control: a no-template control should be included to monitor the contamination.

3. Cross-contamination or mislabeling of barcode primers can lead to the misidentification of samples,

so following precautions are highly recommended when handling barcode primers:

Wear new gloves when working with barcode primers and change gloves if accidentally touch one

barcode primer.

Handle one barcode primer at a time and DO NOT open multiple tubes simultaneously.

Intended Use

For research use only

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Protocol

1. Cell Lysis

1.1 Thaw the Cell Lysis Buffer to room temperature (RT). Vortex and centrifuge the tube briefly at

2000rpm for 3-5 seconds.

1.2 Prepare the following lysis reaction master mix:

Component Volume per Reaction

Cell Lysis Buffer 5μl

Cell Lysis Enzyme 0.5µl

Total Volume 5.5µl

Note: To ensure the best lysis result, make sure that there is no air bubble in the lysis reaction mix.

1.3 Collect biopsy sample(s) into individual 0.2ml PCR tube(s) with 5.5 µl lysis reaction mix and centrifuge

briefly.

Note: Try to minimize the solution carried over with the biopsy sample to 1ul.

To prevent sample loss, DO NOT invert or vortex the tube after sample collection.

(Optional) If biopsy samples are processed immediately, samples can be collected into 5µl Cell Lysis

Buffer and stored at -20°C. 0.5 µl Cell Lysis Enzyme can be added when needed to start the lysis process.

Centrifuge the tube briefly.

Note: To prevent sample loss, make sure that the pipette tip DOES NOT touch the solution when adding the

enzyme. DO NOT invert or vortex the tube after adding the enzyme. Only centrifugation can be

performed.

1.4 Put the PCR tube(s) from step 1.3 in ChromInstTM Sample Prep Station, lock the lid and set up the

lysis program as below:

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1.5 Stop the program as shown below when the lysis process is done. Proceed to the next step

immediately.

2. Pre-Library Preparation

2.1 Thaw the Pre-Lib Buffer to RT. Mix thoroughly and centrifuge briefly.

Figure 1-1：Click the “Lysis” icon to

enter the setup screen

Figure 1-2：Set up the lysis program:

Control mode: “Tube”

Sample volume: “10”

Hotlid control: “On” “105”

Pausing at first seg: “No”

Click “OK” to start the program.

Figure 1-3：Stop the lysis program

The “Remain time” will show “--：--：--”

when the program is complete, click

“Stop” to terminate the program.

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2.2 Prepare the pre-library reaction master mix as follows:


Pre-Lib Buffer 30μl

Pre-Lib Enzyme Mix 1µl

Total Volume 31µl

Mix the reaction thoroughly and centrifuge briefly.

2.3 Add 30μl pre-library reaction mix into each cell lysis product from step 1.5. Mix thoroughly and

centrifuge briefly.

2.4 Incubate the PCR tube(s) from step 2.3 in the ChromInstTM Sample Prep Station, lock the lid and set

up the lysis program as below:

2.5 Stop the program as shown below when the process is done. Proceed to the next step immediately.

Figure 2-1：Click the “Pre_Lib” icon to


Figure 2-2：Set up the Pre_Lib program:



Hotlid control: “On” “105”



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3. Library Preparation

3.1 Thaw the Pre-Lib Buffer to RT. Mix thoroughly and centrifuge briefly.

3.2 Prepare the library reaction master mix as follows:


Library Buffer 30μl

Library Enzyme Mix 0.8µl

Total Volume 30.8µl


3.3 Add 30μl library reaction mix and 1μl Barcode Primer to each pre-library product from step 2.5.


Note: Please refer to “Important Notes” when handling barcode primers.

Use one barcode for each sample and DO NOT use the same barcode for two samples that will

be sequenced in the same sequencing run/lane. Carefully write down the barcode number for

corresponding sample.

3.4 Incubate the PCR tube(s) from step 3.3 in the ChromInstTM Sample Prep Station, lock the lid and

set up the lysis program as below:

Figure 2-3：Stop the Pre_Lib program




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3.5 Stop the program as shown below when the process is done.

Note: If libraries are not sequenced immediately, store the libraries at -20°C. Otherwise, proceed to the

next step.

4. Library Pooling

4.1 Pool equal volume of each library into a new 1.5ml micro-centrifuge tube. For pooling different

numbers of library samples, the volume used for each sample is listed as follows:

Figure 3-1：Click the “LibPrep” icon to


Figure 3-2：Set up the LibPrep program:



Hotlid control: “On”, “105”



Figure 3-3：Stop the LibPrep program




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For example, when pooling two library samples, add 50ul of each sample into the

micro-centrifuge tube.

Note: the remaining library samples can be stored at -20°C.

4.2 Mix the pooled libraries thoroughly and centrifuge briefly.

5. Library Purification

5.1 Beads preparation:

Take out CMPure beads from 2-8°C at least 20 mins before the purification step. Mix CMPure beads

completely by vortexing for 20 seconds. Dispense enough beads for the purification step into a new

1.5ml micro-centrifuge tube and warm beads to RT.

5.2 Add 1 × CMPure beads into pooled libraries. Mix by pipetting up and down >= 10 times and incubate

at RT for 5 min.

For example, add 100μl CMPure bead to 100 μl pooled libraries.

5.3 After incubation, centrifuge the tube briefly and place it on the magnetic stand.

5.4 Wait for about 5 mins or till the solution becomes clear. While keeping the tube on the magnetic

stand, carefully aspirate the solution and discard.

Note: DO NOT touch the beads during this procedure.

5.5 Add 300µl freshly prepared 80% ethanol to the tube. Incubate at RT for 30 seconds and carefully

remove the supernatant.

No. of Library

Samples

Volume of Each

Sample (µl)

No. of Library

Samples

Volume of Each

Sample (µl)

1 50 13 7.7

2 50 14 7.2

3 33.3 15 6.7

4 25 16 6.3

5 20 17 5.9

6 16.7 18 5.6

7 14.3 19 5.3

8 12.5 20 5

9 11.1 21 4.8

10 10 22 4.5

11 9.1 23 4.3

12 8.4 24 4.2

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Note: DO NOT take the tubes off the magnetic stand. DO NOT touch or disrupt the beads during this

procedure.

5.6 Repeat 5.5.

5.7 Remove the ethanol as completely as possible. Air dry the beads on the magnetic stand for about

5-10 mins at RT.

Note: DO NOT over dry the beads.

5.8 Take the tube off the magnetic stand, add 17.5 μl H2O to the tube and resuspend the beads by

vortexing. Centrifuge the tube briefly and incubate at RT for 5 minutes.

5.9 Place the tube onto the magnetic stand and wait till the solution becomes clear. Carefully transfer

15μl supernatant to a new tube.

Note: DO NOT touch the beads during this procedure.

6. Library Quantification

Purified libraries can be quantified using the QubitTM Fluorometer according to manufacturer’s

instructions.

7. Sequencing

Please refer to specific instructions for sequencing on different Illumina next-generation sequencing

platforms (http://www.illumina.com/).

http://www.illumina.com/

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Troubleshooting Guide

Issues Potential Causes Solutions

No or low amplified

product

Sample loss during cell

collection

Redo the cell collection process. Avoid accidentally removal of the genetic material.

Degradation of genome

DNA

Avoid inappropriate storage of cells or template preparation processes that potentially degrade DNA. Decontaminate the work space thoroughly by DNase and RNase decontamination reagents.

Polymerase inhibitors

Polymerase inhibitors carried over from the starting material can cause failure of the WGA reaction. Try to minimize the carryover of buffer to 2µl or less.

Inactive Enzyme

The enzyme should be stored at -20°C. Use cryo-safe cold box to prevent the enzyme from warming when preparing the reaction mix.

Degraded Reagents Minimize freeze/thaw cycling and prepare appropriate aliquots after the first use.

Amplified products in

negative (no-template)

control

Reagents contaminated by

external DNA

Keep kit reagents and the amplified DNA in different storage spaces. Aliquot the reagents after the first use. Use sterile and nuclease-free tubes and filter tips to set up the reactions.

Work area contaminated

by external DNA

Decontaminate the work space thoroughly by DNA and RNA removing reagents

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Technical Support

For technical assistance and additional information, please contact us via:

Phone: +86-21-33190666

Fax：+86- 021-33191962

Web：http://en.yikongenomics.com/

E-mail: [email protected]

Yikon Genomics Co., Ltd.

Room 102, Building I, Hi-Tech Oasis I

No. 888 Tianlin Rd, Caohejing Hi-Tech Park

Shanghai, 200233

China

Phone: +86-21-33190666

Fax: +86- 021-33191962

Email: [email protected]

http://en.yikongenomics.com/

mailto:[email protected]

[email protected]

Documents

For Preimplantation Genetic Screening (PGS) on Illumina ... User Manual.pdf · This kit is used to process embryo biopsy samples for preimplantation genetic screening (PGS) using