May 06, 2026

Targeted DNA Sequencing of LDLR, APOB, PCSK9, and LDLRAP1 Using QIAseq DNA Pro Custom Panel and Illumina MiSeq

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l6jqbkvqe/v1
  • Rano Alieva1,
  • Aleksandr Shek1,
  • Anastasiya Bahachova2,
  • Khurshid Fozilov1,
  • Guzal Abdullaeva1,
  • Alisher Abdullaev3,
  • Lilya Kan1,
  • Shavkat Khoshimov1,
  • Andrey Kim1,
  • Ulugbek Nizamov1,
  • Dilnora Yusupalieva1
  • 1Republican Specialized Scientific and Practical Medical Center for Cardiology, Ministry of Health of the Republic Uzbekistan;
  • 2Independent Consultant, Tbilisi, Georgia;
  • 3Center for Advanced Technologies, Ministry of Higher Education, Science and Innovation of the Republic Uzbekistan, Tashkent, Uzbekistan
Anastasiya Bahachova
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6jqbkvqe/v1
Protocol Citation: Rano Alieva, Aleksandr Shek, Anastasiya Bahachova, Khurshid Fozilov, Guzal Abdullaeva, Alisher Abdullaev, Lilya Kan, Shavkat Khoshimov, Andrey Kim, Ulugbek Nizamov, Dilnora Yusupalieva 2026. Targeted DNA Sequencing of LDLR, APOB, PCSK9, and LDLRAP1 Using QIAseq DNA Pro Custom Panel and Illumina MiSeq. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6jqbkvqe/v1
Manuscript citation:
Alieva R, Shek AB, Bahachova AV, Fozilov KG, Abdullaeva GJ, Abdullaev AA, Kan LE, Khoshimov SU, Kim AR, Nizamov UI, Yusupalieva DB.

Genetic Yield of Next-Generation Sequencing for Detecting Monogenic Familial Hypercholesterolemia in Uzbek Patients with Coronary Artery Disease.

Manuscript under review.
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 04, 2026
Last Modified: May 06, 2026
Protocol  Integer ID: 316296
Keywords: targeted dna sequencing of ldlr, targeted dna sequencing, qiaseq targeted dna pro panel, ldlrap1 using qiaseq dna pro custom panel, ldlrap1 gene, targeted dna pro panel, detection of genetic variant, genetic variant, ldlr, illumina miseq platform, using qiaseq, illumina miseq this protocol
Disclaimer
This protocol is based on manufacturer-recommended workflows and user manuals, with steps adapted and applied according to the design of a custom QIAseq Targeted DNA Pro panel.
Abstract
This protocol describes targeted next-generation sequencing (NGS) for the detection of genetic variants in LDLR, APOB, PCSK9, and LDLRAP1 genes using QIAseq Targeted DNA Pro panels and the Illumina MiSeq platform.
Attachments
Icon for Supplementary Table 1 – Amplicon design for QIAseq Targeted DNA Pro panel.pdf
Supplementary Table ...
608KB
Guidelines
Target Panel Design and Coverage

The custom QIAseq Targeted DNA Pro panel (QIAGEN) is designed to capture coding exons and flanking intronic regions (including splice sites) of the LDLR, APOB, PCSK9, and LDLRAP1 genes. Target enrichment is performed using a multiplex PCR-based approach with pooled gene-specific primers. Target regions are defined based on RefSeq transcripts (GRCh38 reference genome). The panel consists of overlapping amplicons to ensure comprehensive coverage of all target regions. Each region is covered by one or more primer pairs to minimize dropout and ensure uniform coverage. Amplicons span coding regions as well as flanking intronic sequences, enabling detection of variants affecting splice sites. Primer design includes both forward and reverse orientations and is optimized to improve amplification efficiency across GC-rich and structurally complex regions.

The custom panel contains 236 primers and covers 22,388 bp of target regions, with 100% ROI coverage according to the manufacturer’s design summary.
Materials
General equipment, reagents, and consumables
Thermomixer or heating block (56°C)
Multichannel pipettes
Single-channel pipettes
Nuclease-free pipette tips and tubes
Microcentrifuge (for 1.5–2 ml tubes)
Microcentrifuge tubes (1.5 ml or 2 ml) 
Vortex mixer
Ethanol (80–100%) Nuclease-free water Ice

Equipment, reagents, and consumables for DNA extraction
DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany)

Equipment, reagents and consumables for DNA quantification and quality assessment

Quantus Fluorometer (Promega, Madison, WI, USA)
QuantiFluor ONE dsDNA System (Promega, Madison, WI, USA) 0.5ml PCR Tubes (Promega, Madison, WI, USA) NanoPhotometer NP80 spectrophotometer (Implen, Munich, Germany)
Equipment, reagents, and consumables for library preparation
QIAseq Targeted DNA Pro Custom (96) Panel (QIAGEN, Hilden, Germany) QIAseq Targeted DNA Pro UDI Set B (96) (QIAGEN, Hilden, Germany) DNA LoBind tubes, 1.5 mL (Eppendorf, Germany) PCR tubes and caps (0.2 mL) or 96-well PCR plates DNA LoBind tubes, 1.5 mL (Eppendorf, cat. no. 022431021)
C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA).
Quantus Fluorometer (Promega, Madison, WI, USA)
QuantiFluor ONE dsDNA System (Promega, Madison, WI, USA) DynaMag-96 Side Magnet (Thermo Fisher Scientific, USA)
Equipment, reagents, and consumables for sequencing
MiSeq Reagent Kit v2 (300 cycles) (Illumina, San Diego, CA, USA)
MiSeq System (Illumina, San Diego, CA, USA)
PhiX Control v3 (Illumina, San Diego, CA, USA)
Local Run Manager (LRM) software (Illumina, San Diego, CA, USA)
Software and computational resources

CLC Genomics Workbench (QIAGEN, Aarhus, Denmark)
Safety warnings
  • Handle NaOH with care, as it is a corrosive chemical. Wear appropriate personal protective equipment, including gloves and eye protection.
  • Avoid cross-contamination between samples, especially during library preparation and indexing steps. Use filter tips and change tips between samples.
  • Ensure accurate pipetting when preparing libraries, as errors may lead to uneven library representation or sequencing bias.
  • Do not disturb magnetic beads during cleanup steps, as this may result in loss of DNA.
  • Avoid over-drying magnetic beads, which can reduce DNA recovery efficiency.
  • Use freshly prepared reagents where indicated (e.g., NaOH) to ensure optimal performance.
  • Improper library loading concentration may result in low cluster density or overclustering on the MiSeq flow cell.
Before start
  • This protocol requires a custom QIAseq Targeted DNA Pro panel. The panel design is provided by the manufacturer and should not be modified unless revalidated.
  • Ensure that the primer design and genomic coordinates correspond to the GRCh38 reference genome.
  • Detailed amplicon design, including genomic coordinates and primer sequences, is provided in Supplementary Table 1 (see Attachments). Users should review the panel design prior to starting the protocol.
DNA Extraction
Extract genomic DNA from peripheral blood samples using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions.
DNA Quantification
Quantify DNA using the QuantiFluor ONE dsDNA System (Promega, Madison, WI, USA) in combination with the Quantus Fluorometer (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

1. Prepare the QuantiFluor ONE dsDNA dye working solution according to the manufacturer’s instructions.
2. Prepare standards by adding 1 µL of DNA standard to 200 µL of dye working solution.

For each sample, prepare assay tubes as follows:
Add 200 µL of QuantiFluor ONE dye working solution
Add 1–2 µL of DNA sample

3. Incubate the reactions for 5 minutes at room temperature, protected from light.
4. Measure fluorescence using the Quantus Fluorometer according to the dsDNA assay protocol, including calibration with blank and standards.
5. Record DNA concentration values for each sample. If necessary, dilute samples to ensure that measurements fall within the assay’s linear range and repeat the measurement.

Notes:
  • DNA concentration is determined using a fluorometric method specific for double-stranded DNA.
  • Measurements should fall within the linear range of the assay.
  • Measurements may be performed in duplicate where possible.

Library Preparation
Prepare sequencing libraries using the QIAseq Targeted DNA Pro Kit (QIAGEN) and the QIAseq Targeted DNA Pro UDI Set B (96) (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions.

General considerations for library preparation:
  • Perform all enzymatic reaction setup steps on ice unless otherwise specified.
  • Avoid vortexing enzymatic reactions.
  • When preparing multiple samples, prepare a master mix with an additional 5–10% volume.

DNA Fragmentation and end prepare
1. Use 10–80 ng of genomic DNA as input.
2. Prepare the fragmentation and end-preparation reaction mix as shown in Table 1.
AB
ComponentVolume per reaction
Genomic DNA Variable (10–80 ng)
10× FX Buffer 1.4 µL
5× WGS FX Mix 2.8 µL
Nuclease-free water To 14 µL total
Total volume 14 µL
Table 1. Reaction mix for DNA fragmentation and end preparation (standard genomic DNA)
3. Mix thoroughly by pipetting.
4. Incubate the reaction using the conditions shown in Table 2.
ABC
StepTemperatureTime
1 4 1 min
2 32 10 min
3 65 15 min
4 4 Hold
Table 2. Incubation conditions for fragmentation and end preparation
5. After incubation, place samples on ice and proceed immediately to adapter ligation.

Adapter Ligation

1. Use the entire fragmentation/end-prep reaction (~14 µL) as input.
2. Prepare the adapter ligation reaction mix according to Table 3.
AB
ComponentVolume per reaction (µL)
Fragmentation/end-prep reaction (input DNA) 14
2.5× UPH Ligation Buffer 10
AdP-IL5 Phased Adapter 1.5
DNA Ligase 2.5
Final reaction volume 28
Table 3. Reaction mix for adapter ligation
3. Mix thoroughly by pipetting.
4. Incubate the reaction using the conditions shown in Table 4.
ABC
StepTemperatureTime
1 4°C 1 min
2 20°C 15 min
3 65°C 20 min
4 4°C Hold
Table 4. Incubation conditions for adapter ligation

Notes:
  • Do not use a heated lid during the 20°C incubation step. Alternatively, set the lid temperature to ≤65°C.
  • After incubation, place samples on ice and proceed immediately to ligation cleanup.
Ligation Cleanup

1. Use the entire adapter ligation reaction (~28 µL) as input.
2. Add 2 µL of Ligation Cleanup Reagent to each reaction.
3. Mix thoroughly by pipetting and briefly centrifuge.
4. Incubate using the conditions shown in Table 5.
ABC
StepTemperatureTime
1 4°C 1 min
2 37°C 15 min
3 95°C 10 min
4 4°C Hold
Table 5. Incubation conditions for ligation cleanup
5. After incubation, place samples on ice and proceed to target enrichment.
Target Enrichment (PCR)

  1. Use the entire ligation cleanup product (~30 µL) as input.
  2. Prepare the target enrichment PCR reaction mix as shown in Table 6.
AB
ComponentVolume per reaction (µL)
Cleaned adapter-ligated DNA (input) 30
5× TE-PCR Buffer 10
QIAseq Targeted DNA Pro Custom Panel 6.3
SmP-IL5 TE-PCR-F Primer 2
TE-PCR Modifier 1.95
DNA Polymerase 2.4
Final reaction volume 52.65
Table 6. Reaction mix for target enrichment PCR
3. Add the reaction mix to the cleaned adapter-ligated DNA.
4. Mix thoroughly by pipetting and briefly centrifuge.
5. Place tubes or plate in a thermal cycler with a heated lid (>100°C).
6. Perform target enrichment PCR using the cycling conditions in Table 7.

Important:
  • Because the custom panel contained fewer than 12,000 primer entries per tube, cycling conditions recommended for panels with <12,000 primers/tube were applied.

ABC
StepTimeTemperature
Initial denaturation 2 min 98°C
8 cycles 15 s 2 min 98°C 65°C
1 cycle 3 min 72°C
Hold ∞ 4°C
Table 7. Cycling conditions for target enrichment PCR (panel with <12,000 primers/tube)

7. After PCR, allow the thermal cycler to return to 4°C.
8. Place samples on ice and proceed immediately to TE-PCR cleanup.

Notes:
  • Annealing and extension times may vary depending on panel complexity.
  • A ramp rate of ~2°C/s is recommended for optimal amplification performance.

Optional:
  • Samples may be stored at −20°C for up to several days prior to cleanup if necessary.
TE-PCR Cleanup

1. Transfer samples to ice immediately after completion of target enrichment PCR.
2. Mix each reaction gently and transfer 20 µL of the target enrichment PCR product to a new PCR tube or plate.
3. Add 5 µL of TE-PCR Cleanup Reagent to each sample.
4. Mix thoroughly by pipetting and briefly centrifuge.
5. Place tubes or plate into the thermal cycler with a heated lid (>100°C).
6. Perform TE-PCR cleanup using the incubation conditions shown in Table 8.
ABC
Step Temperature Time
1 4°C 1 min
2 37°C 15 min
3 95°C 10 min
4 4°C Hold
Table 8. Incubation conditions for TE-PCR cleanup
7. Upon completion, allow the thermal cycler to return to 4°C.
8. Place samples on ice and proceed immediately to universal PCR.

Notes:
  • Formation of visible precipitate during this step is expected and does not affect downstream amplification.
  • Keep samples on ice prior to the next step.
Universal PCR (Indexing)

Important points before starting:
  • Use a unique UDI primer pair for each sample to prevent index cross-contamination.
  • Use a fresh pipette tip for each well when handling the UDI plate.
  • Do not reuse wells once the foil seal has been pierced.
  • Avoid cross-contamination between index wells.
  • Each. well of the QIAseq Targeted DNA Pro UDI Set B plate contains a unique pair of dual indices (i5 and i7). The plate is organized in a standard 96-well format (A–H × 1–12), where each well corresponds to one unique index combination. Assign one well per sample to ensure unique dual indexing across the sequencing run
Figure 1. Layout of UDI index plate (Set B, 96-well)

1. Use 25 µL of the TE-PCR cleanup product as input.
2. Prepare the universal PCR reaction mix as shown in Table 9.
AB
ComponentVolume per reaction (µL)
Cleaned TE-PCR product 25
5× UPCR Buffer 20
UDI primer pair (from UDI Set B plate) 5
DNA Polymerase 4.8
Nuclease-free water 45.2
Final reaction volume 100
Table 9. Reaction mix for universal PCR (indexing)

3. Add the reaction mix to the cleaned TE-PCR product.
4. Add 5 µL of a unique UDI primer pair from the QIAseq Targeted DNA Pro UDI Set B plate to each reaction.
5. Mix thoroughly by pipetting and briefly centrifuge.
6. Place tubes or plate in a thermal cycler with a heated lid (>100°C).
7. Perform universal PCR using the cycling conditions shown in Table 10.
ABC
Step TemperatureTime
Initial denaturation 98°C 2 min
29 cycles 98°C 62°C 15 s 30 s
Final extension 72°C 3 min
Hold 4°C ∞
Table 10. Cycling conditions for universal PCR

8. Upon completion, allow the thermal cycler to return to 4°C.
9. Proceed immediately to post-PCR cleanup using magnetic beads.
Post-PCR Cleanup (Magnetic Bead Purification)

Important points before starting:
  • Bring QIAseq beads to homogeneity before use by gentle mixing.
  • Perform all bead binding, washing, and elution steps at room temperature.
1. Use the entire universal 2. PCR reaction (100 µL) as input.
3. Add 80 µL of QIAseq beads to each reaction.
4. Mix thoroughly by pipetting until homogeneous.
5. Incubate for 5 minutes at room temperature.
6. Place tubes or plate on a magnetic stand until the solution clears.
7. Carefully remove and discard the supernatant without disturbing the beads.
8. Remove the tubes or plate from the magnetic stand.
9. Add 100 µL of nuclease-free water followed by 80 µL of QIAseq Bead Binding Buffer.
10. Mix thoroughly by pipetting.
11. Return the tubes or plate to the magnetic stand and allow the solution to clear.
12. Carefully remove and discard the supernatant.
13. Add 200 µL of 80% ethanol to wash the beads.
14. Remove and discard the ethanol.
15. Repeat the ethanol wash once.
16. Ensure complete removal of residual ethanol after the second wash.
17. Air-dry the beads at room temperature for ~10 minutes until dry.

Important:
  • Avoid over-drying; however, slight over-drying does not significantly affect elution.

18. Remove the tubes or plate from the magnetic stand.
19. Elute DNA by adding 30 µL of nuclease-free water.
20. Mix thoroughly and incubate briefly.
21. Place on the magnetic stand until the solution clears.
22. Transfer ~28 µL of the eluate to a clean tube or plate.
23. Proceed to library quantification and quality control.

Optional:
  • Libraries may be stored at −30°C to −15°C prior to sequencing.
Library normalization and pooling
1. Measure library concentrations (ng/µL) as described in Section 2.
2. Use mass concentration as a proxy for library molarity, assuming relatively uniform amplicon sizes for the targeted panel design.
3. Calculate the required dilution for each library based on measured concentration.
4. Dilute each library individually with nuclease-free water to approximately 2–4 nM, or to comparable concentrations across samples.
5. Combine equal volumes of normalized libraries into a single tube to generate the pooled library.
6. Mix the pooled library thoroughly by pipetting.

Notes:
  • Libraries are normalized to ensure equal mass input from each sample.
  • Equal-volume pooling provides a practical approximation of equimolar representation.
  • This approach is appropriate for targeted panels with relatively uniform amplicon sizes.

Output:
  • The pooled library is ready for downstream dilution, denaturation, and sequencing on the Illumina MiSeq platform.
Library Denaturation, Dilution, and Loading
1. Use the pooled library obtained from the previous section for denaturation.
2. Combine 5 µL of pooled library with 5 µL of freshly prepared 0.2 N NaOH.
3. Mix thoroughly and incubate at room temperature for 5 minutes.
4. Add 990 µL of pre-chilled HT1 buffer to the denatured library to obtain a 20 pM library.
5. Dilute the 20 pM library with pre-chilled HT1 buffer to the desired loading concentration.
6. Adjust the final loading concentration to 4–7 pM based on fluorometric quantification.
7. Prepare the PhiX control library according to Illumina recommendations.
8. Add PhiX control to the library pool at ~10% (v/v) to increase sequence diversity.
9. Load the prepared library onto the Illumina MiSeq system according to the manufacturer’s instructions.
10. Create a sample sheet using Local Run Manager (LRM) v2 according to the Illumina workflow.
11. Configure the sequencing run with the following parameters:
  • Read 1: 149 cycles
  • Read 2: 149 cycles
  • Index 1 (i7): 10 cycles
  • Index 2 (i5): 10 cycles
12. Start the sequencing run using Local Run Manager (LRM) v2 according to the manufacturer’s instructions.
Protocol references
QIAGEN. QIAseq Targeted DNA Pro Handbook. 2024.
https://www.qiagen.com/en-us/resources/download/KitHandbook/hb-2979-003-hb-qiaseq-targeted-dna-pro-illumina-0724-ww

QIAGEN. DNeasy Blood & Tissue Handbook. 2023.
https://www.qiagen.com/en-us/resources/download/kithandbook/hb-2061-004-hb-dny-blood-tissue-0623-ww

Promega. Quantus Fluorometer Operating Manual.
https://worldwide.promega.com/-/media/files/resources/protocols/technical-manuals/101/quantus-fluorometer-operating-manual.pdf

Illumina. MiSeq System Denature and Dilute Libraries Guide.
https://support-docs.illumina.com/IN/MiSeq_DnD/Content/MiSeq/DnD-MiSeq.htm

QIAGEN Digital Insights. CLC Genomics Workbench User Manual.
https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/User_Manual.pdf

QIAGEN Digital Insights. Biomedical Genomics Analysis Plugin.
https://digitalinsights.qiagen.com/plugins/biomedical-genomics-analysis/