Illumina TruSeq Bovine Parentage Reference Guide

Chapter 1: Overview

Introduction

This protocol explains how to prepare up to 96 uniquely indexed paired-end libraries of genomic DNA (gDNA) using the Illumina® TruSeq® Bovine Parentage Kit. The kit supports sequencing targeted regions of the bovine genome with up to 273 amplicons in a single multiplex reaction. This highly targeted approach enables a wide range of applications for discovering, validating, and screening genetic variants in a rapid and efficient manner.

The TruSeq Bovine Parentage library prep protocol offers:

• Multiplexing capability to amplify up to 273 amplicons in a single reaction and sequence up to 96 samples in a single sequencing run.
• Fast and simple workflow to generate up to 273 amplicons across 96 samples within a single plate with less than 3 hours hands-on time.
• Streamlined 96-well based workflow amenable to automation.

The TruSeq Bovine Parentage library prep supports:

• Project creation and management using the Illumina online DesignStudio software for 273 amplicons and the UMD 3.1 bovine reference genome.
• Automated data analysis to perform variant calling and analysis across all samples using simple on-instrument, automated analysis software.
• Secondary analysis to convert variant calls to genotype calls using the Sequence Genotyper.
• The convenience of a fully integrated DNA-to-data solution including online ordering, assay, sequencing, automated data analysis, and offline software for reviewing results.

DNA Input Recommendations

Quantify the starting genomic material using a fluorescence-based quantification method, such as a Qubit dsDNA Assay Kit or PicoGreen, rather than a UV-spectrometer-based method. Fluorescence-based methods, which employ a double-stranded DNA (dsDNA) specific dye, specifically and accurately quantify dsDNA even in the presence of many common contaminants. In contrast, UV spectrometer methods based on 260 OD readings are prone to overestimating DNA concentrations due to the presence of RNA and other contaminants commonly found in genomic DNA (gDNA) preparations.

If necessary, dilute gDNA so that the concentration is between 10–25 ng/µl.

Assessing DNA Quality

The ratio of absorbance at 260 nm to absorbance at 280 nm is used as an indication of sample purity. This protocol is optimized for DNA with absorbance ratio values of 1.8–2.0.

Additional Resources

Visit the TruSeq Bovine Parentage kit support page on the Illumina website for documentation, software downloads, training resources, and information about compatible Illumina products.

The following documentation is available for download from the Illumina website:

• Custom Protocol Selector: A wizard for generating customized end-to-end documentation that is tailored to the library prep method, run parameters, and analysis method used for the sequencing run. Access at support.illumina.com/custom-protocol-selector.html.
• TruSeq Bovine Parentage Protocol Guide (document # 1000000012391): Provides instructions for the experienced user.
• TruSeq Bovine Parentage Checklist (document # 1000000012394): Provides a checklist of steps for the experienced user.
• TruSeq Bovine Parentage Consumables & Equipment List (document # 1000000012395): Provides an interactive checklist of user-provided consumables and equipment.
• Sequence Genotyper Software Guide: Provides information about using the Sequence Genotyper software and available analysis options.
• TruSeq Amplicon Quick Reference Card: Provides information about using the Illumina® Experiment Manager (IEM) to create and edit sample sheets compatible with your Illumina sequencing system and analysis software.

For additional setup information, see the Questions & Answers section of the TruSeq Bovine Parentage support page.

Chapter 2: Protocol

Introduction

This chapter describes the TruSeq Bovine Parentage protocol.

• Follow the protocols in the order shown, using the specified volumes and incubation parameters.
• Review Best Practices from the TruSeq Bovine Parentage support page on the Illumina website.

Prepare for Pooling

If you plan to pool libraries, record information about your samples before beginning library prep. Different methods are available depending on the sequencing instrument you are using. See the TruSeq Bovine Parentage Kit support page for more information.

Tips and Techniques

Unless a safe stopping point is specified in the protocol, proceed immediately to the next step.

Avoiding Cross-Contamination

• When adding or transferring samples, change tips between each sample.
• When adding adapters or primers, change tips between each row and each column.
• Remove unused index adapter tubes from the working area.

Sealing the Plate

• Always seal the 96-well plate before the following steps in the protocol: Shaking steps, Vortexing steps, Centrifuge steps, Thermal cycling steps.
• Apply the adhesive seal to cover the plate and seal with a rubber roller.
• Microseal 'B' adhesive seals are effective at -40°C to 110°C, and suitable for skirted or semiskirted PCR plates. Use Microseal 'B' for shaking, centrifuging, and long-term storage.
• Microseal 'A' adhesive film is effective for thermal cycling and easy to cut when using fewer than 96 wells.

Plate Transfers

• When transferring volumes between plates, transfer the specified volume from each well of a plate to the corresponding well of the other plate.

Centrifugation

• Centrifuge at any step in the procedure to consolidate liquid or beads in the bottom of the well, and to prevent sample loss.

Handling Beads

• Pipette bead suspension slowly.
• When mixing, mix thoroughly.
• If beads are aspirated into the pipette tips, dispense back to the plate on the magnetic stand and wait until the liquid is clear (~2 minutes).
• When washing beads: Use the appropriate magnet for the plate. Dispense liquid so that beads on the side of the wells are wetted. Keep the plate on the magnet until the instructions specify to remove it. Do not agitate the plate while on the magnetic stand. Do not disturb the bead pellet.

TruSeq Bovine Parentage Workflow

The following figure illustrates the TruSeq Bovine Parentage workflow. Safe stopping points are marked between steps.

Figure 1: TruSeq Bovine Parentage Workflow

The workflow consists of 7 main steps:

1. Hybridize Oligo Pool: Hands-on: 15 minutes, Total: 1 hour 35 minutes. Reagents: BVP1, OHS2, ACD1, ACP1.
2. Remove Unbound Oligos: Hands-on: 20 minutes. Reagents: ELM4, SW1, UB1.
3. Extend and Ligate Bound Oligos: Hands-on: 5 minutes, Total: 50 minutes. Reagents: ELM4.
4. Amplify Libraries: Hands-on: 30 minutes, Total: 1 hour 55 minutes - 2 hours 15 minutes. Reagents: PMM2, TDP1, NaOH, Index i5, Index i7.
5. Clean Up Libraries: Hands-on: 20 minutes, Total: 50 minutes. Reagents: EBT, AMPure XP beads, EtOH.
6. Normalize Libraries: Hands-on: 30 minutes, Total: 1 hour 20 minutes. Reagents: LNA1, LNB1, LNW1, LNS2, NaOH.
7. Pool Libraries: Hands-on: 10 minutes. Reagents: HT1.

Optional Overnight Incubation is noted between steps 4 and 5. Safe Stopping Point markers are indicated between steps 6 and 7, and after step 6.

Hybridize Oligo Pool

This process hybridizes a custom oligo pool that contains upstream and downstream oligos specific to your targeted regions of interest. Perform replicates to increase confidence in somatic variant calls.

Consumables

• BVP1 (Bovine Parentage Oligo Tube)
• OHS2 (Oligo Hybridization for Sequencing 2)
• ACD1 (Amplicon Control DNA)
• ACP1 (Control Oligo Pool)
• HYP (Hybridization Plate) barcode label
• Diluted high-quality gDNA
• 96-well PCR plate
• Adhesive aluminum foil seal
• Microseal 'B' adhesive seals

WARNING: This set of reagents contains formamide, an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including eye protection, gloves, and laboratory coat. Handle used reagents as chemical waste and discard in accordance with the governmental safety standards for your region. For environmental, health, and safety information, see the SDS for this kit at support.illumina.com/sds.html.

About Reagents

• OHS2: Aspirate and dispense slowly due to the viscosity of the reagent. Before each use, vortex thoroughly and then centrifuge briefly. Make sure that all precipitates have dissolved. When mixing, mix thoroughly.
• ACD1 and ACP1: Include ACD1 and ACP1 in every batch of samples being prepared. Use of these controls enables Illumina Technical Support to troubleshoot if you need assistance. If these controls are excluded from your assay, assistance will not be provided.
• Do not mix BVP1 and OHS2 for storage. If combined, BVP1 becomes unstable even when stored frozen.

Remove Unbound Oligos

This process removes unbound oligos from genomic DNA using a size-selection filter. Two wash steps using SW1 ensure complete removal of unbound oligos. A third wash step using UB1 removes residual SW1 and prepares samples for the next step.

Consumables and Equipment

• ELM4 (Extension-ligation Mix 4)
• SW1 (Stringent Wash 1)
• UB1 (Universal Buffer 1)
• Filter plate with lid
• Adapter roller
• Midi plate

WARNING: This set of reagents contains formamide, an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including eye protection, gloves, and laboratory coat. Handle used reagents as chemical waste and discard in accordance with the governmental safety standards for your region. For environmental, health, and safety information, see the SDS for this kit at support.illumina.com/sds.html.

Preparation

Prepare the following consumables:

Procedure

1. Make sure that the heat block has cooled to 40°C and the HYP plate seal is secure.
2. Remove from the heat block.
3. Centrifuge at 1000 × g for 1 minute.
4. Transfer each sample to the corresponding well of the FPU plate.
5. Cover and centrifuge at 2400 × g for 2 minutes.
6. Wash 2 times as follows: Add 45 µl SW1 to each sample well. Cover and centrifuge at 2400 × g for 2 minutes. If SW1 does not drain completely, centrifuge again for up to 10 minutes.
7. Discard flow-through.

Figure 2: FPU Assembly

The Filter Plate Unit (FPU) is assembled from top to bottom. Components include:

• A: Lid
• B: Filter plate
• C: Adapter collar
• D: Midi plate

Label the completed assembly FPU. Wash the wells to be used in the assay as follows. Use new wells only. Add 45 µl SW1 to each well. Cover the FPU plate. Centrifuge at 2400 × g for 10 minutes. If a significant amount (> 15 µl/well) of residual buffer remains in multiple wells (≥ 10 wells/plate), switch to a new filter plate.

Continue procedure: Reassemble the FPU plate for continued use. Add 45 µl UB1 to each sample well. Cover and centrifuge at 2400 × g for 2 minutes. If UB1 does not drain completely, centrifuge again for up to 10 minutes.

Extend and Ligate Bound Oligos

This step connects the hybridized upstream and downstream oligos. A DNA polymerase extends from the upstream oligo through the targeted region, followed by ligation to the 5' end of the downstream oligo using a DNA ligase. The result is the formation of products containing the targeted regions of interest flanked by sequences required for amplification.

Consumables

• ELM4 (Extension-Ligation Mix 4)
• Foil adhesive seal

Procedure

1. Add 45 µl ELM4 to each sample well of the FPU plate.
2. Apply the seal and incubate at 37°C for 45 minutes.
3. During incubation, proceed to the next step.

Amplify Libraries

This step amplifies the extension-ligation products and adds index 1 (i7) adapters, index 2 (i5) adapters, and sequences required for cluster formation.

Consumables

• PMM2 (PCR Master Mix 2)
• Index i5 adapters (A5XX)
• Index i7 adapters (A7XX)
• TDP1 (TruSeq DNA Polymerase 1)
• 50 mM NaOH (less than one week old; prepared from 10 N NaOH)
• 96-well skirted PCR plate
• IAP (Indexed Amplification Plate) barcode label
• Microseal 'A' adhesive film
• Microseal 'B' adhesive seal (for 2°C to 8°C storage)

About Reagents

• PMM2/TDP1: Combine PMM2 and TDP1 immediately before use. Do not combine and store the combined PMM2/TDP1 mixture. When mixing, mix thoroughly.

Preparation

Prepare the following consumables:

Prepare fresh 50 mM NaOH. Save the following PCR program on a thermal cycler: 95°C for 3 minutes; 25 cycles of: 95°C for 30 seconds, 66°C for 30 seconds, 72°C for 60 seconds; 72°C for 5 minutes; Hold at 10°C. Apply the IAP label to a new 96-well PCR plate.

Procedure

1. Arrange the Index 1 (i7) adapters in columns 1–12 of the TruSeq Index Plate Fixture.
2. Arrange the Index 2 (i5) adapters in rows A–H of the TruSeq Index Plate Fixture.

Figure 3: TruSeq Index Plate Fixture

The fixture holds index adapters. Labels indicate:

• A: Rows A–H: Index 2 (i5) adapters (white caps)
• B: Columns 1–12: Index (i7) adapters (orange caps)
• C: IAP plate

Place the plate on a TruSeq Index Plate Fixture. Using a multichannel pipette, add 4 µl of each Index 1 (i7) adapter down each column. Replace the cap on each i7 adapter tube with a new orange cap. Using a multichannel pipette, add 4 µl of each Index 2 (i5) adapter across each row. Replace the cap on each i5 adapter tube with a new white cap. Remove the FPU plate from the incubator and do the following: Replace the aluminum foil seal with the filter plate lid. Centrifuge at 2400 × g for 2 minutes. Add 25 µl 50 mM NaOH to each well. Pipette to mix. Incubate at room temperature for 5 minutes. Add 56 µl TDP1 to a full tube (2.8 ml) of PMM2. Invert to mix. Transfer 22 µl PMM2/TDP1 mixture to each well of the IAP plate. Transfer eluted samples from the FPU plate to the IAP plate as follows: Using fine tips, pipette to mix the NaOH in the first column of the FPU plate. Transfer 20 µl NaOH to the corresponding column of the IAP plate. Pipette to mix. Transfer remaining columns from the FPU to the IAP plate. Discard the waste collection midi plate.

NOTE: Set aside the metal adapter collar for future use. If you partially used an FPU plate, mark the used wells and store the FPU plate and lid in a sealed plastic bag.

Apply Microseal 'A' and centrifuge at 1000 × g for 1 minute. Transfer the IAP plate to the post-amplification area. Place on the preprogrammed thermal cycler and run the PCR program.

SAFE STOPPING POINT: If you are stopping, seal the plate and store at 2°C to 8°C for up to 2 days. Alternatively, leave on the thermal cycler overnight.

Clean Up Libraries

This step uses AMPure XP beads to purify the PCR products from other reaction components.

Consumables and Equipment

• EBT (Elution Buffer with Tris)
• AMPure XP beads
• Barcode labels
• CLP (Cleanup Plate)
• LNP (Library Normalization Plate)
• 96-well midi plates (2)
• Microseal 'B' adhesive seals
• Freshly prepared 80% ethanol (EtOH)
• Magnetic stand-96

Preparation

Prepare the following consumables:

Prepare 40 ml (for 96 samples) fresh 80% ethanol from 100% ethanol. Apply the CLP barcode label to a new midi plate. Apply the LNP barcode label to a new midi plate.

Procedure

1. Centrifuge the IAP plate at 1000 × g for 1 minute.
2. [Optional] Run an aliquot of the library and control using any of the following methods: 5 µl on 4% agarose gel; 1 µl on an Agilent Bioanalyzer using a DNA 1000 chip; 2 µl on an Advanced Analytical Fragment Analyzer using the Standard Sensitivity NGS Fragment Analysis Kit. Expect the PCR product size to be 200–300 bp.

NOTE: Assess library quality by gel electrophoresis, Fragment Analyzer, or Bioanalyzer for oligo pools being used for the first time. You do not need to assess the quality of every sample in the experiment. To enable Illumina Technical Support to troubleshoot if you need assistance, assess the quality of the control reaction generated with the ACD1 and ACP1. Process the control reaction using the same conditions as BVP1.

Figure 4: Agarose Gel Example

Shows DNA bands. Label A points to the Expected ACP1/ACDI PCR product (~350 bp). Label B points to Primers.

Figure 5: Bioanalyzer Example (ACP1)

Shows a chromatogram. Label A indicates a Marker, Label B indicates the Expected ACP1/ACDI PCR product for 250bp amplicons (~350bp), and Label C indicates a Marker.

Figure 6: Bioanalyzer Example (BVP1)

Shows a chromatogram. Label A indicates a Marker, Label B indicates the Expected BVP1/gDNA PCR product (~250 bp), and Label C indicates a Marker.

Add 60 µl AMPure XP beads to each well of the CLP plate. Transfer all the supernatant from each well of the IAP plate to the corresponding well of the CLP plate. Apply the seal and shake at 1800 rpm for 2 minutes. Incubate at room temperature for 10 minutes. Place on a magnetic stand and wait until the liquid is clear (~2 minutes). Remove and discard all supernatant from each well. Wash 2 times as follows: Add 200 µl of 80% EtOH to each sample well. Incubate on the magnetic stand for 30 seconds. Remove and discard all supernatant from each well. Use a 20 µl pipette to remove residual EtOH from each well. Remove from the magnetic stand and air-dry for 10 minutes. Add 30 µl EBT to each well. Apply the seal and shake at 1800 rpm for 2 minutes. Make sure that all beads are resuspended. If necessary, pipette to mix and repeat the shaking step. Incubate at room temperature for 2 minutes. Place on a magnetic stand and wait until the liquid is clear (~2 minutes). Transfer 20 µl supernatant from each well of the CLP plate to the corresponding well of the LNP plate. Apply the seal and centrifuge at 1000 × g for 1 minute.

Normalize Libraries

This step normalizes the quantity of each library for balanced representation in pooled libraries. Only samples containing DNA require processing through the subsequent steps.

Consumables and Equipment

• LNA1 (Library Normalization Additives 1)
• LNB1 (Library Normalization Beads 1)
• LNW1 (Library Normalization Wash 1)
• LNS2 (Library Normalization Storage buffer 2)
• SGP (Storage Plate) barcode label
• 0.1 N NaOH (freshly prepared)
• 96-well PCR plate, skirted
• 15 ml conical tube
• Microseal 'B' adhesive seals
• Magnetic stand-96 (use with midi 96-well storage plates)

WARNING: This set of reagents contains formamide, an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including eye protection, gloves, and laboratory coat. Handle used reagents as chemical waste and discard in accordance with the governmental safety standards for your region. For environmental, health, and safety information, see the SDS for this kit at support.illumina.com/sds.html.

WARNING: This set of reagents contains β-mercaptoethanol. Perform the following procedure in a hood or well-ventilated area.

About Reagents

• Use a P1000 pipette to transfer LNB1 to LNA1.
• When mixing, mix thoroughly.
• Mix only the amounts of LNA1 and LNB1 required for the current experiment.
• Store remaining LNA1 and LNB1 separately at their respective temperatures.
• Make sure that LNB1 is resuspended before use. Homogeneous resuspension is essential for consistent cluster density on the flow cell.

Preparation

Prepare the following consumables:

Prepare fresh 0.1 N NaOH. Label a new 96-well plate SGP.

Procedure

1. For 96 samples, add 4.4 ml LNA1 to a new 15 ml conical tube.
2. Use a P1000 pipette to resuspend LNB1.
3. Transfer 800 µl LNB1 to the 15 ml conical tube of LNA1. Invert to mix.
4. Add 45 µl LNA1/LNB1 to each well of the LNP plate.
5. Apply the seal and shake at 1800 rpm for 30 minutes. Durations other than 30 minutes can affect library representation and cluster density.
6. Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
7. Remove and discard all supernatant.
8. Remove from the magnetic stand.
9. Wash 2 times as follows: Add 45 µl LNW1 to each library well. Apply the seal and shake at 1800 rpm for 5 minutes. Place on a magnetic stand and wait until the liquid is clear (~2 minutes). Remove and discard all supernatant.
10. Use a 20 µl pipette to remove residual LNW1 from each well.
11. Remove from the magnetic stand.
12. Add 30 µl fresh 0.1 N NaOH to each well.
13. Apply the seal and shake at 1800 rpm for 5 minutes.
14. If the libraries are not resuspended, pipette to mix, and then shake at 1800 rpm for 5 minutes.
15. Place the LNP plate on a magnetic stand and wait until the liquid is clear (~2 minutes).
16. Add 30 µl LNS2 to each well of the SGP plate.
17. Transfer 30 µl supernatant from each well of the LNP plate to the corresponding well of the SGP plate. Pipette to mix.
18. Apply the seal and centrifuge at 1000 × g for 1 minute.

SAFE STOPPING POINT: If you are stopping, seal the plate and store at -25°C to -15°C for up to 30 days.

Pool Libraries

Pooling libraries combines equal volumes of normalized libraries in a single tube. After pooling, dilute and denature the library pool before loading libraries for the sequencing run.

Consumables

• PAL (Pooled Amplicon Library) barcode label
• LoBind microcentrifuge tube
• RNase/DNase-free 8-tube strips and caps

About Reagents

• Store the PAL tube at -25°C to -15°C for later use.

Preparation

1. If the SGP plate was stored frozen, prepare as follows: Thaw at room temperature. Centrifuge at 1000 × g for 1 minute. Pipette to mix.
2. To prepare for the sequencing run, begin thawing reagents according to the instructions for your instrument.
3. Label a new Eppendorf tube PAL.

Procedure

1. Centrifuge at 1000 × g for 1 minute.
2. Transfer 5 µl of each library to an 8-tube strip, column by column. Seal the plate and store at -25°C to -15°C.
3. Transfer the contents of the 8-tube strip to the PAL tube. Pipette to mix.
4. Denature and dilute pooled libraries to the loading concentration for the instrument you are using. See the denature and dilute libraries guide for your instrument.

Appendix A: Supporting Information

Introduction

The protocols described in this guide assume that you have reviewed the contents of this appendix, confirmed your kit contents, and obtained all the required consumables and equipment.

Acronyms

Kit Contents

Make sure that you have all the reagents identified in this section before proceeding to the library preparation procedures. The TruSeq Custom Amplicon Index Kit is included in the TruSeq Bovine Parentage Kit.

TruSeq Bovine Parentage Kit Contents (20004795)

Box 1 - Store in the Pre-PCR Area

This box also contains the HYP, FPU, and IAP barcode labels.

WARNING: This set of reagents contains formamide, an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including eye protection, gloves, and laboratory coat. Handle used reagents as chemical waste and discard in accordance with the governmental safety standards for your region. For environmental, health, and safety information, see the SDS for this kit at support.illumina.com/sds.html.

Box 2 - Store in the Pre-PCR Area

This box is shipped at room temperature. As soon as you receive your kit, remove LNB1 from box 2 and store at 2°C to 8°C in the post-amplification area. Keep the filter plate in the pre-PCR area at room temperature.

Box 3 - Store in the Post-PCR Area

This box also contains the CLP, LNP, SGP, PAL, DAL barcode labels.

TruSeq Custom Amplicon Index Kit Contents

Box 1 - Store in Pre-PCR Area

Box 2 - Store in Pre-PCR Area

Additional Components

Consumables and Equipment

Make sure that you have the required user-supplied consumables and equipment before starting the protocol.

NOTE: Use a dedicated set of consumables and equipment for pre-PCR and post-PCR procedures. The TruSeq Bovine Parentage library prep protocol requires different magnetic stands for pre-PCR and post-PCR procedures.

Consumables

Pre-PCR Equipment

Post-PCR Equipment

Thermal Cyclers

Use the following recommended settings for selected thermal cycler models. Before performing library prep, validate any thermal cyclers not listed.

Index Sequences

The TruSeq Custom Amplicon Index Kit contains the following indexed adapter sequences.

i7 Index Adapter

i5 Index Adapter

Technical Assistance

For technical assistance, contact Illumina Technical Support.

Table 1: Illumina General Contact Information

Table 2: Illumina Customer Support Telephone Numbers

Safety data sheets (SDSs) are available on the Illumina website at support.illumina.com/sds.html.

Product documentation is available for download in PDF from the Illumina website. Go to support.illumina.com, select a product, then select Documentation & Literature.