Enhanced HLA Typing Resolution per Celera AlleleSEQR® Motif-specific Sequencing Primers

Jana Kobrle, Robert Bruce, Birgit Drews, Douglas A. Bost Celera, Alameda, California, USA

AbstractAim: In a renewed effort to enhance the resolution power of Celera’s AlleleSEQR® HLA-SBT products, we initiated a project to design additional sequencing primers to resolve ambiguous HLA allele combinations.

Methods: Active market surveillance was conducted to identify ambiguities that could not be resolved with the currently available products. Reported allele combinations containing at least two common, well documented (CWD) alleles were analyzed using the HLA/IMGT database sequence alignment tool and searched for common cis/trans ambiguities across all loci. The results were applied to designing a set of new motif-specific ambiguity primers (MSPs), which were subsequently tested using the 3100, 3130, 3730 and 3500 platforms.

Results: The extended panel of MSPs was designed to utilize the smallest number of sequencing primers to resolve the maximum number of ambiguities across all class I loci by targeting common motifs rather than locus-specific sequences. Half of the reported class I ambiguities could be resolved with only 5 new motif-specific ambiguity primers functioning across multiple class I loci. Resolution for class I loci could be increased through an additional 9 primers targeting more locus-specific motifs. Five primers were designed for class II. We further tested the use of new MSPs with adjusted run conditions to allow sequencing though introns. Using this approach on a subset of reported allele combinations, together with novel designs to improve resolution in the proximity of primer annealing site, we were able to successfully resolve 90% of reported ambiguities.

Conclusions: We designed a new panel of motif-specific sequencing primers, which will increase the overall number of Celera AlleleSEQR HLA-SBT ambiguity resolving sequencing primers to a total of 53 for class I and class II HLA genes. This expanded panel of reagents is estimated to resolve the majority of allele combinations currently encountered in clinical labs.

Evaluation of Ambiguous Allele CombinationsA list of ambiguous alleles reported by the labs to be unresolvable using the current menu of commercially available AlleleSEQR® sequencing reagents was reviewed for potential resolving strategies using the HLA/IMGT database sequence alignment tool. The results of this evaluation are summarized in Table 1. Approximately 20% of ambiguities were determined to be unresolvable due to technical limitations, where targeted resolving ambiguity is found either too close to the primer annealing site (gap < 40) or is in a different exon than the primer annealing site and read-length of at least 400 bp is required for full resolution.

Table 1. The feasibility of resolution by sequencing primers targeting only one of the present alleles was evaluated with sequencing parameters set as follows: read length < 400 bp and gap of 40 bp. Gap is defined as a distance between ambiguity and position of 3’-end of a primer annealing site.

Locus Reported ambiguitites Resolvable Required read

length > 400bp Gap < 40 bp

HLA-A 22 16 2 4

HLA-B 36 23 4 + 1 partial 8

HLA-C 45 42 1 + 1 partial 1 + 1 partial

HLA-DRB1 18* 12 - 5

HLA-DQB1 15 12 - -

* One DRB1 ambiguity will be rsolvable by the updated DRB1 kit covering the end of exon 2

Primer DesignThe extended panel was designed to utilize the smallest number of sequencing primers to resolve the maximum number of ambiguities across all class I loci by targeting common motifs rather than locus-specific sequences. Half of reported class I ambiguities could be resolved with only 5 new motif-specific ambiguity primers functioning across multiple class I loci. Resolution for class I loci could be increased through an additional 7 primers targeting more locus-specific motifs. Two more primers targeting only a small number of ambiguities from the list were added due to high frequencies of given allele combinations. Five primers were designed for class II, enabling unambiguous resolution of 73% of reported class I and class II combinations. Based on additional customers’ feedback, DPB1 ambiguity resolution primer annealing at codon 8 was added into the class II set. In order to further increase their resolution capabilities, 19 out 20 newly designed primers were modified to achieve better coverage in the proximity of their 3’-prime end.

Table 2: Description of new Motif-specific Sequencing Primers (MSPs). The importance of each primer is expressed as number of potential resolutions (number of reviewed allele combinations resolvable by given primer) and by unique resolution (number of reviewed allele combinations resolvable only by given primer).

MSP ID MSP ID Tested Loci* Exon Position Nucleotide Direction Potential

ResolutionsUnique

ResolutionsCurrent Status

MSP-1.26 MSP1.01 C 2 201 a F 6 2 in production

MSP-1.27 MSP1.02 A 2 282 g R 7 5 in production

MSP-1.28 MSP1.03 B 2 292 t R 4 4 in production

MSP-1.29 MSP1.04 B, C 2 302 g R 11 8 in production

MSP-1.30 MSP1.05 A, C 2 341 c R 11 6 in production

MSP-1.31 MSP1.06 B, C 3 355 c F 11 5 in production

MSP-1.32 MSP1.07 C 3 368 a F 16 5 in production

MSP-1.33 MSP1.08 A, C 3 527 a R 11 3 in production

MSP-1.34 MSP1.09 A, C 3 538 t R 12 4 in production

MSP-1.35 MSP1.10 A 3 570 g R 3 3 in production

MSP-1.36 MSP1.11 B 3 572 c R 4 4 in production

MSP-1.37 MSP1.12 B 3 583 t R 8 5 in production

MSP-1.38 MSP1.13 A 3 385 c F 1 1 in production

MSP-1.39 MSP1.14 B 2 97 c F 1 1 in production

MSP-2.11 MSP2.01 DQB1 2 122 a F 7 4 in production

MSP-2.12 MSP2.02 DQB1 2 353 c R 8 5 in production

MSP-2.13 MSP2.03 DRB1 2 164 a F 3 3 in production

MSP-2.14 MSP2.04 DRB1 2 258 t F 5 5 in production

MSP-2.15 MSP2.05 DRB1 2 189 g R 3 3 in production

MSP-2.16 MSP2.06 DPB1 2 111 t F NA NA in production

*The specificity of MSPs is not limited to tested loci.

Materials and MethodsSamples

Two sets of sixteen previously typed DNA samples were selected for testing class I and class II MSPs from panels supplied through the UCLA Immunogenetics Center. All DNAs were diluted to 20 ng/µl, prior to the start of the experiment.

PCR Amplification and Purification

PCR amplification, enzymatic clean-up and dilution of PCR products were performed according to the AlleleSEQR HLA-SBT protocol.

Sequencing workflow using MSPs

Preparation of MSP sequencing mixes:

MSP sequencing mixes were prepared according to the following table using 20 µM MSP, SeqMix 1 (List #09K58-03; Abbott Molecular) and water.

Number of Reactions 5 10 15 20 25 30Volume of 20 µM MSP (µL) 1 2 3 4 5 6

Volume of SeqMix1 (µL) 30 60 90 120 150 180Volume of water (µL) 9 18 27 36 45 54

*To ensure sufficient reaction volume per number of samples (N), master mix for N + 1 or N + 2 samples was prepared.

Sequencing reactions

1. 8 µL of MSP sequencing mix was added to each reaction tube.

2. 2 µL of diluted ExoSAP-IT treated PCR product was added to the appropriate sequencing reaction mix. Reactions were centrifuged briefly to combine.

3. The following thermal cycling profile was run:

Number of Cycles Temperature Time

2596 20 seconds50 30 seconds60 2 minutes

1 4 Infinite

Ethanol Precipitation and Preparation for Loading

1. 2 µL of NaOAc/EDTA Buffer was added to each sequencing reaction.

2. Tubes were centrifuged briefly to ensure the NaOAc/EDTA buffer was properly combined with the sequencing reactions (5 seconds at 500 X g).

3. 25 µL of absolute EtOH was added to each sequencing reaction, vortexed briefly but vigorously.

4. Reactions were centrifuged at 2000 X g for 30 minutes.

5. The supernatant was removed by inverting the tubes/tray onto a paper towel. Inverted tubes/tray were centrifuged at 50-100 X g for 10 seconds.

6. 50 µL of 80% EtOH was added to each sequencing reaction and centrifuged at 2000 X g for 5 minutes.

7. Step 5 was repeated.

8. Sequencing reactions were prepared for loading by resuspending in 15 µL of HiDi® Formamide, centrifuged briefly.

9. Reactions were denatured in a thermal cycler for 2 minutes at 95°C.

10. A) Standard data collection was performed according to the following specifi-cations:

Mobility Files and Run Modules

Instrument Mobility File Run ModuleInjection

ParametersCollection

Parameters

AB 3100 KB _3100POP6 BDTv1.mob RapidSeq36_POP6_1 1.0-1.5kV 10 sec. 1800 sec.

AB 3130 KB_3130_POP6_BDTv1.mob RapidSeq36_POP6_1 1.0-1.5kV 10 sec. 1800 sec.

AB 3730 KB_3730_POP7_BDTv1.mob RapidSeq36_POP7_1 1.0-1.5kV 10 sec. 1800 sec.

AB 3500xL KB_3500_POP6_BDTv1.mob RapidSeq50_POP6XL_E 1.6kV 10 sec. 2100 sec.

10. B) Read-lengths above 400 bp were obtained using following run modules with increased collection times:

Mobility Files and Run Modules for Long Read Lengths

Instrument Mobility File Run ModuleInjection

ParametersCollection

Parameters

AB 3500xL KB_3500_POP6_BDTv1.mob StdSeq50_POP6xl 1.6kV 18 sec. 5653 sec.

ResultsNew MSPs testing

All MSPs were tested on 16 UCLA samples that have 0, 1, and 2 alleles that match the specificity of each primer resulting in homozygous, heterozygous or no sequence. Class I and class II DNA panels were designed to include a minimum of five samples with homozygous outcome. The analysis was done using SBTengine Software (GenDx). Figures 1–3 show data for locus A, B and C generated by core sequencing primers and MSP1.13, MSP1.03 and MSP1.09, respectively.

Figure 1. A*01:01:01:01 + A*03:01:01:01, exon 3, homozygous sequence generated using MSP1.13.

Figure 2. B*07:02:01 + B*35:01:01:01, exon 2, homozygous sequence generated using MSP1.03.

Figure 3. C*02:02:02 + C*08:02:01, exon 3, homozygous sequence generated using MSP1.09.

Improved resolution in the proximity of a primer annealing site:

The distance between the 3’-prime end of a sequencing primer and a first readable nucleotide depends on the method used for purification of sequencing reactions and on the type of polymer used for sequencing. Nineteen out of twenty new MSPs were modified to decrease this gap by approximately 20 bp. This parameter can be set in SBTengine in the GSSP tab in the preferences menu. Allele resolution within 10 bp from the primer annealing site using DRB1 specific MSP2.05 is shown on Figure 4.

Figure 4: MSP2.05 (3’ primer position: 189) resolving DRB1 alleles within 10 bp of primer annealing site (positions 196, 197 and 199).

Additional resolution by sequencing through intron:

Modification of data collection specifications to generate longer sequencing reads allowed for resolution of ambiguous positions located more than 400 bp away from primer annealing site, requiring sequencing through the intron. Figure 5 depicts MSP1.08 annealing at position 527 in exon 3 and resolving HLA B alleles in exon 2 at positions 272–322 (500bp read length required, fig. 5a) and at positions 142–167 (630 bp read length required, Figure 5b) by sequencing through the 245 bp long intron sequence (not shown).

Figure 5a: Primer MSP 1.08 resolving HLA B alleles at positions 272–322 in exon 2 by sequencing through intron

Figure 5b: Primer MSP 1.08 resolving HLA B alleles at positions 142–167 in exon 2 by sequencing through intron

Contact email: Jana.Kobrle@celera.com

Table 3. Table of selected class I allele combinations resolvable using new motif-specific sequencing primers (read length < 400bp and gap of 40bp).

A*01:01:01:01 A*03:01:01:01 = A*03:97 A*36:04A*01:01:01:01 A*11:01:01 = A*11:39 A*36:04A*03:01:01:01 A*11:01:01 = A*03:65 A*11:24:02 or A*03:69N A*11:24:01A*03:01:01:01 A*11:01:01 = A*03:65 A*11:24:02 or A*03:127 A*11:20A*03:01:01:01 A*32:01:01 = A*03:08 A*32:17A*03:01:01:01 A*74:01 = A*03:08 A*74:13

A*23:01:01 A*24:03:01 = A*23:04 A*24:02:01:01A*23:01:01 A*29:01:01:01 = A*23:13 A*29:20A*25:01:01 A*66:02 = A*25:02 A*66:03A*26:01:01 A*66:02 = A*26:13 A*66:03A*26:01:01 A*68:01:01 = A*26:13 A*68:03:03A*26:01:01 A*68:01:02 = A*26:13 A*68:03:01 or A*66:05 A*68:84A*26:01:01 A*68:02:01:01 = A*26:13 A*68:31

A*66:01 A*68:03:01 = A*66:05 A*68:01:02A*66:02 A*68:03:01 = A*66:03 A*68:01:02

B*07:02:01 B*44:02:01:01 = B*07:114 B*44:22B*07:02:01 B*15:01:01:01 = B*07:114 B*15:125B*07:02:01 B*35:01:01:01 = B*07:114 B*35:54B*07:02:01 B*38:01:01 = B*07:91 B*38:14B*07:02:01 B*44:03:01 = B*07:114 B*44:105B*13:02:01 B*51:01:01 = B*13:15 B*51:02:01B*14:02:01 B*38:01:01 = B*14:18 B*38:14

B*15:01:01:01 B*18:01:01 = B*15:38:01 B*18:11 or B*15:225 B*18:43B*15:03:01 B*37:01:01 = B*15:123 B*37:13B*18:01:01 B*35:01:01 = B*18:11 B*35:24:01B*18:01:01 B*37:01:01 = B*18:11 B*37:04:01B*18:01:01 B*39:01:01:01 = B*18:11 B*39:48

B*35:01:01:01 B*44:02:01:01 = B*35:137 B*44:84B*35:01:01:01 B*44:02:01:01 = B*35:71 B*44:91 or B*35:137 B*44:84B*35:01:01:01 B*44:03:01 = B*35:71 B*44:37B*35:01:01:01 B*39:11 = B*35:08:01 B*39:05:01B*40:06:01:01 B*51:01:01 = B*40:148, B*51:09

B*44:03 B*50:01:01 B*44:46 B*49:02B*45:01 B*50:01:01 = B*45:04 B*50:02B*45:01 B*52:01:01:01 = B*45:04 B*52:15

B*49:01:01 B*52:01:01:01 = B*49:09 B*52:11B*51:01:01 B*56:01:01 = B*51:09:01 B*56:25C*01:02:01 C*02:02:02 = C*01:48 C*02:08C*01:02:01 C*06:02:01:01 = C*01:09 C*06:06C*01:02:01 C*07:01:01 = C*01:17 C*07:40 or C*01:58 C*07:16C*01:02:01 C*12:03:01:01 = C*01:09 C*12:24C*02:02:02 C*03:04:01:01 = C*02:27:01, C*03:07C*02:02:02 C*04:01:01:01 = C*02:32 C*04:94C*02:02:02 C*07:01:01 = C*02:13 C*07:165C*02:02:02 C*08:02:01 = C*02:32 C*08:29C*03:03:01 C*07:01:01 = C*03:11:01 C*07:20 or C*03:67 C*07:26C*03:03:01 C*07:02:01:01 = C*03:18 C*07:127C*03:03:01 C*15:02:01 = C*03:04:01:01 C*15:12 or C*03:31 C*15:17

C*03:04:01:01 C*04:01:01:01 = C*03:98 C*04:85C*03:04:01:01 C*04:03:01 = C*03:32 C*04:107C*03:04:01:01 C*07:02:01:01 = C*03:41 C*07:133C*03:04:01:01 C*15:02:01 = C*03:07 C*15:07C*03:04:01:01 C*15:02:01 = C*03:07 C*15:07 or C*03:08 C*15:17C*04:01:01:01 C*06:02:01:01 = C*04:54 C*06:09 or C*04:94:01 C*06:06C*04:01:01:01 C*07:01:01 = C*04:61 C*07:165C*04:01:01:01 C*12:02:01 = C*04:15:01 C*12:10:01 or C*12:10:02 C*12:10:02C*04:01:01:01 C*12:03:01:01 = C*04:94:01 C*12:24C*04:01:01:01 C*14:02:01 = C*04:29 C*14:12 or C*04:33 C*14:13C*04:01:01:01 C*16:01:01 = C*04:15:02 C*16:23

C*04:04:01 C*07:01:01 = C*04:13 C*07:165C*05:01:01:01 C*07:02:01:01 = C*05:23 C*07:17C*05:01:01:01 C*12:03:01:01 = C*05:08 C*12:24 or C*08:02:01 C*12:04:02C*05:01:01:01 C*16:01:01 = C*08:02:01 C*16:02:01C*06:02:01:01 C*07:02:01:01 = C*06:11 C*07:76C*06:02:01:01 C*14:02:01 = C*06:06 C*14:16

C*07:01:01 C*15:02:01 = C*07:165 C*15:31C*07:02:01:01 C*08:02:01 = C*07:17 C*08:07C*07:02:01:01 C*12:03:01:01 = C*07:27 C*12:11C*07:02:01:01 C*15:05:01 = C*07:10 C*15:22

C*07:04:01 C*08:02:01 = C*07:12 C*08:07C*08:02:01 C*12:03:01:01 = C*08:29 C*12:24C*08:02:01 C*16:01:01 = C*08:12 C*16:07

C*12:03:01:01 C*14:02:01 = C*12:24 C*14:16C*14:02:01 C*16:01:01 = C*14:05 C*16:23

Table 4. Table of selected class II allele combinations resolvable using new motif-specific sequencing primers (read length < 400bp and gap of 40bp).

DRB1*01:01:01 DRB1*07:01:01 = DRB1*01:21 DRB1*07:11

DRB1*03:01:01:01 DRB1*13:01:01 = DRB1*03:07 DRB1*13:27 or DRB1*03:15 DRB1*13:61:02 or DRB1*03:19 DRB1*13:20

DRB1*03:01:01:01 DRB1*14:01:01 = DRB1*03:06 DRB1*14:35 or DRB1*03:07 DRB1*14:82

DRB1*04:01:01 DRB1*04:05:01 = DRB1*11:04:01 DRB1*04:09

DRB1*11:04:01 DRB1*14:01:01 = DRB1*11:54 DRB1*14:32 or DRB1*11:56 DRB1*14:35

DRB1*03:01:01:01 DRB1*11:01:01 = DRB1*03:05:01 DRB1*11:04:01 or DRB1*03:14 DRB1*11:84:02 or DRB1*03:15 DRB1*11:27:02

DQB1*03:02:01 DQB1*04:02:01 = DQB1*03:05:03 DQB1*04:03 :02

DQB1*03:02:01 DQB1*06:03:01 = DQB1*03:08 DQB1*06:32

DQB1*04:02:01 DQB1*06:02:01 = DQB1*04:03:01 DQB1*06:23

DQB1*06:02:01 DQB1*06:03:01 = DQB1*06:11 :02 DQB1*06:14:02

DQB1*06:02:01 DQB1*06:04:01 = DQB1*06:03:01 DQB1*06:22 or DQB1*06:08:02 DQB1*06:15 or DQB1*06:09 DQB1*06:14:02

DQB1*06:03:01 DQB1*06:04:01 = DQB1*06:07:02 DQB1*06:08:01 or DQB1*06:39 DQB1*06:41 or DQB1*06:21 DQB1*06:32

DQB1*03:02:01 DQB1*06:02:01 = DQB1*03:08 DQB1*06:48

DQB1*03:03:02 DQB1*06:02:01 = DQB1*03:30 DQB1*06:11:01

Conclusions We recently assessed a list of ambiguous allele combinations

reported by HLA labs as unresolvable by currently available

sequence resolution primers. Based on this input we designed

a new panel of motif-specific sequencing primers, which

increased the overall number of Celera AlleleSEQR HLA-SBT

sequencing primers to a total of 55 for class I and class

II ambiguity resolution. This expanded panel of reagents

is estimated to resolve the majority of allele combinations

currently encountered in clinical labs.

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