NeXtype is a software aiming at high-accuracy HLA typing based on short amplicon Next Generation Sequencing (NGS) data from Illumina MiSeq systems.
NeXtype employs advanced statistical algorithms ...that use high number of reads and associated Q-values delivered by NGS. In our NGS setup, MiSeq provides raw sequence data of paired-end reads. For all samples, 6 loci are considered on exons 2 and 3. The neXtype workflow is organized in several consecutive steps. Up-front, pre-run data is assembled, including a tree-structured HLA library and primer information. In the first step of an individual typing run, the primer sequence is identified for each read by a multi-level process that involves direct string comparison and also adapts to sequencing errors. Next, for each pair of reads the most likely matches in the HLA library are determined using Q-values and the predefined tree structure. The third step evaluates the linkage between all matches of each locus and exon and orders them by significance. The fourth step classifies the called alleles as results or discards them as crossover products, statistical noise, co-amplifications etc. For each locus, allele pairs are obtained by combining the respective exon information. Although neXtype reliably rates its results, a user can override the result classification if deemed necessary and declare results as artifacts for future reference.
NGS results by neXtype have successfully been cross-validated with Sanger sequencing results of 6840 loci. In average, 98.7% of all typings have high resolution. 94.9% do not require user interaction, 2.7% require user editing for improvement of the result, and in 2.4 % data quality was insufficient to allow HLA typing. This high degree of automation reduces analysis time to less than 2 h per 2000 loci.
NeXtype matches the needs of high throughput HLA labs providing accurate typing with minimal user intervention. Currently, 9,000 loci are typed per day.
For HLA donor centers/registries the cost of HLA typing currently is a major constriction limiting the resolution and number of loci typed at registration. Therefore, we aimed to set up a workflow ...for HLA typing by NGS with the clear focus on cost efficiency and high throughput.
The workflow is based on direct sequencing of short amplicons on Illumina MiSeq instruments by 3 simple steps. Step 1: PCR amplification of exons 2 and 3 of HLA loci A, B, C, DRB1, DQB1 and DPB1 on Fluidigm Access Array chips. Adapters for the NGS sequencing reaction and tags to identify the samples are incorporated during PCR. Step 2: PCR products of 48 samples are pooled and purified by SPRIselect. Step 3: 8 purified sample pools (384 samples) are combined, denatured and sequenced on an Illumina MiSeq sequencer (2 x 250bp). All steps are automated on liquid handling systems equipped with barcode scanners that communicate with a custom LIMS for sample tracking to prevent sample mix-up by design. For HLA allele assignment we developed the neXtype software featuring high-quality typing in combination with minimal user intervention.figure1
We have carried out typing of more than 200,000 samples at 6 loci since entering routine operation in January 2013. Turn around time is less than 14 days. Despite our focus on increasing cost efficiency, we achieve a typing resolution that exceeds the current standard of Sanger typing as applied for donor registration.
High throughput routine operation confirms that NGS measures up to the high expectations. The described workflow for HLA typing by NGS has proven simpler, faster, better and more cost effective than Sanger sequencing.
Next Generation Sequencing (NGS) has entered routine in HLA sequencing laboratories. We implemented a short amplicon-based NGS workflow for high-throughput donor registry HLA typing of 6 loci at high ...resolution. An elaborate primer design was key for this cost-efficient approach.
Potential primer binding sites are restricted by the read lengths of NGS systems. For most amplicons this requires a mix of primers that has to be optimized for uniform amplification of all alleles. In our case primers were designed to amplify under identical PCR conditions for all amplicons to facilitate the use of the 48.48 Fluidigm Access Array. Using the Illumina MiSeq 2x250bp system we chose primer binding sites to achieve full exon coverage with an overlap of 50 to 200 bases of exons 2 and 3 for HLA loci A, B, C, DRB1, DQB1 and DPB1. Self-developed Perl and Python scripts were used for analysis of each primer’s performance, as individual NGS reads can be classified and quantified. Primer sets were assessed for the ratio of target reads between amplicons and the ratio of target reads to off-target reads. Off-target reads were classified in three categories. Uniformity of amplification was assessed with the minimum requirement of a 1:5 ratio of target reads between alleles in a heterozygous sample. Based on this approach over 700 primers were screened and a final set of 66 primers was chosen.
In three MiSeq runs 1140 samples (6840 loci) were typed and compared with previous Sanger sequencing results. All typings were in full concordance. In average 80.2% of the allocated reads corresponded to target HLA alleles, with a range from 63.9% (HLA-B exon 2) to 89.2% (HLA-A exon 2).
The validated primer set was launched in December 2012 and yields 98.7% high resolution typing. In an automated workflow, this approach allows very cost-efficient high-throughput (currently about 1500 donors per day) HLA donor registry typing.
Aim Typing of potential donors for all transplantation relevant factors at registration can speed up donor selection which benefits certain patients. Despite several studies regarding the effect of ...the KIR repertoire on the outcome of hematopoietic stem cell transplantation (HSCT), KIR typing data is commonly not available for donor selection – to some extent due to the costs of conventional KIR typing methods. Our amplicon-based NGS typing approach has reduced HLA typing costs considerably. Here, we report a method for cost effective high-throughput KIR typing that enables the addition of KIR typing to the standard profile for all newly registered donors. Methods Exons 4, 5 and 7 are amplified in three PCR reactions using primer mixes targeting all KIR genes. The PCR products contain sample specific identification sequences and can therefore be combined for joint sequencing on Illumina MiSeq or HiSeq instruments. Up to 4800 samples are sequenced on one 2 × 250 rapid run HiSeq flowcell yielding on average about 60,000 reads per sample evenly split between HLA and KIR. Sequencing data is analyzed by neXtype, an inhouse software, and currently yields presence/absence calls for the KIR genes with KIR2DS4 and KIR2DS4N being distinguished and KIR2DL5A and KIR2DL5B being combined into KIR2DL5. Results Validation yielded 100% concordance with the pretypings for all 109 samples passing the predefined internal quality criteria. Within the first 4 months in 2015 we performed successful KIR typing for more than 100.000 samples. Initial analysis indicates that haplotypes lacking the KIR core genes 3DP1 and 2DL4 are more common than previously anticipated. Conclusion Our amplicon-based NGS typing approach enables us to type up to 5000 samples/day for KIR. Typing costs including KIR increased only moderately compared to the former profile including 6 HLA loci (A, B, C, DRB1, DQB1 and DPB1), CCR5, and blood groups ABO and Rh. This facilitated DKMS to expand the profile for all newly registered donors to include KIR typing.
Aim We describe 2127 new HLA alleles: 598 HLA-A, 755 HLA-B and 774 HLA-C alleles, accounting for 28.9 % of the currently known HLA class I alleles. Methods Buccal swabs or blood samples of newly ...recruited donors from three national DKMS donor centers were typed for HLA using sequencing-based typing (SBT) at the ASHI-accredited laboratory of HistoGenetics (Ossining, NY). New HLA class I alleles were identified after determination of DNA sequences of HLA exons 2 and 3. Comparison of the new allele sequences and their most homologous counterpart lead to detection and description of nucleotide variations in DNA sequences. Self-assessed parentage of individuals carrying new HLA class I alleles were examined. Results Of the 2127 new HLA class I alleles described, 1336 (62.8%) were observed in donors from Germany, 422 (19.8%) in US donors, 213 (10.0%) in donors from Poland, and 156 (7.3%) new alleles were found in donors from at least two countries. We identified new alleles in 57 allele groups, including 192 C∗ 07, 144 A∗ 02 and 112 C∗ 03 alleles, The majority (93.8%) of the new alleles corresponded to single nucleotide variations. 67.0% of the new alleles comprised non-synonymous nucleotide substitutions and 30.0% silent mutations. The remaining 3.0% showed nonsense mutations identified in null alleles. New alleles were found disproportionally often in minority donors. Further, 527 new alleles (24.8%) were identified in multiple individuals. Conclusions We described 2127 new HLA class I alleles that have been identified in upfront HLA typing of newly registered DKMS stem cell donors. The occurrence of many new alleles in multiple individuals who originated partly from different populations indicates that newly identified alleles are not necessarily rare and thus may be of relevance for actual stem cell donor searches.
LBOR01 Lange, Vinzenz; Boehme, Irina; Paul, Patrick ...
Human immunology,
04/2015, Volume:
76, Issue:
4
Journal Article
Peer reviewed
Aim DKMS Life Science Lab adopted an NGS based HLA typing workflow in early 2013. Meanwhile more than a million samples have been successfully typed by NGS forming the basis for this review of the ...chances and pitfalls of such an approach. Methods Our NGS workflow is based on direct sequencing of PCR amplified exons on Illumina MiSeq instruments. Exons 2 and 3 of HLA-A, B, C, DRB1, DQB1 and DPB1 are amplified by PCR on Fluidigm Access Array chips. Recently the profile was extended to cover in addition ABO, Rhesus and CCR5. Amplicons of up to 768 samples are pooled for a sequencing run. Sequencing data is analysed by the in-house developed software neXtype. Results The rather simple workflow proved to be very robust. Loading of the Fluidigm chips is critical and, due to the low volumes, needs careful optimization and regular maintenance of the involved liquid handlers. PCR continuously yielded sufficient product for loading on the sequencers. The MiSeqs in general produce very high quality sequence data. Low diversity samples like amplicon products pose a particular challenge to the MiSeqs but are meanwhile handled well. However, care must be taken to keep the rate of very short PCR artefacts low as those may negatively impact the overall quality. For analysis of the data care must be taken to identify crossover PCR products which may resemble true HLA alleles. In addition, due to PCR artefacts the rate of error at a particular position might be significantly higher than indicated by the q -values. When taking this into account software like neXtype can provide highly automated and accurate results at a fraction of the time of Sanger data analysis. Given adequate software we identified imbalanced amplification as the main source of error. Rigorous quality control of every reagent batch with regard to even amplification is essential. In addition samples with DNA concentrations below 3 ng/μl should not be applied to 192 Fluidigm chips to avoid random loss of alleles. Conclusions Switching to NGS as the primary tool for HLA typing was highly beneficial for the lab. The NGS based workflow has proven superior to the previous Sanger based workflow in every aspect: Costs, hands on time, resolution, turnaround time, stability and scalability. We conclude that NGS technology has matured to a point where it is appropriate for everyday routine operation.
Aim Next Generation Sequencing (NGS) has entered routine in HLA sequencing laboratories. We implemented a short amplicon-based NGS workflow for high-throughput donor registry HLA typing of 6 loci at ...high resolution. An elaborate primer design was key for this cost-efficient approach. Methods Potential primer binding sites are restricted by the read lengths of NGS systems. For most amplicons this requires a mix of primers that has to be optimized for uniform amplification of all alleles. In our case primers were designed to amplify under identical PCR conditions for all amplicons to facilitate the use of the 48.48 Fluidigm Access Array. Using the Illumina MiSeq 2x250bp system we chose primer binding sites to achieve full exon coverage with an overlap of 50 to 200 bases of exons 2 and 3 for HLA loci A, B, C, DRB1, DQB1 and DPB1. Self-developed Perl and Python scripts were used for analysis of each primer’s performance, as individual NGS reads can be classified and quantified. Primer sets were assessed for the ratio of target reads between amplicons and the ratio of target reads to off-target reads. Off-target reads were classified in three categories. Uniformity of amplification was assessed with the minimum requirement of a 1:5 ratio of target reads between alleles in a heterozygous sample. Based on this approach over 700 primers were screened and a final set of 66 primers was chosen. Results In three MiSeq runs 1140 samples (6840 loci) were typed and compared with previous Sanger sequencing results. All typings were in full concordance. In average 80.2% of the allocated reads corresponded to target HLA alleles, with a range from 63.9% (HLA-B exon 2) to 89.2% (HLA-A exon 2). Conclusions The validated primer set was launched in December 2012 and yields 98.7% high resolution typing. In an automated workflow, this approach allows very cost-efficient high-throughput (currently about 1500 donors per day) HLA donor registry typing.
124-P Lange, Vinzenz; Boehme, Irina; Paul, Patrick ...
Human immunology,
11/2013, Volume:
74
Journal Article
Peer reviewed
Aim For HLA donor centers/registries the cost of HLA typing currently is a major constriction limiting the resolution and number of loci typed at registration. Therefore, we aimed to set up a ...workflow for HLA typing by NGS with the clear focus on cost efficiency and high throughput. Methods The workflow is based on direct sequencing of short amplicons on Illumina MiSeq instruments by 3 simple steps. Step 1: PCR amplification of exons 2 and 3 of HLA loci A, B, C, DRB1, DQB1 and DPB1 on Fluidigm Access Array chips. Adapters for the NGS sequencing reaction and tags to identify the samples are incorporated during PCR. Step 2: PCR products of 48 samples are pooled and purified by SPRIselect. Step 3: 8 purified sample pools (384 samples) are combined, denatured and sequenced on an Illumina MiSeq sequencer (2 x 250bp). All steps are automated on liquid handling systems equipped with barcode scanners that communicate with a custom LIMS for sample tracking to prevent sample mix-up by design. For HLA allele assignment we developed the neXtype software featuring high-quality typing in combination with minimal user intervention.figure1 Results We have carried out typing of more than 200,000 samples at 6 loci since entering routine operation in January 2013. Turn around time is less than 14 days. Despite our focus on increasing cost efficiency, we achieve a typing resolution that exceeds the current standard of Sanger typing as applied for donor registration. Conclusions High throughput routine operation confirms that NGS measures up to the high expectations. The described workflow for HLA typing by NGS has proven simpler, faster, better and more cost effective than Sanger sequencing.
160-P Sauter, Jürgen; Hofmann, Jan A; Lange, Vinzenz ...
Human immunology,
11/2013, Volume:
74
Journal Article
Peer reviewed
Aim NeXtype is a software aiming at high-accuracy HLA typing based on short amplicon Next Generation Sequencing (NGS) data from Illumina MiSeq systems. Methods NeXtype employs advanced statistical ...algorithms that use high number of reads and associated Q-values delivered by NGS. In our NGS setup, MiSeq provides raw sequence data of paired-end reads. For all samples, 6 loci are considered on exons 2 and 3. The neXtype workflow is organized in several consecutive steps. Up-front, pre-run data is assembled, including a tree-structured HLA library and primer information. In the first step of an individual typing run, the primer sequence is identified for each read by a multi-level process that involves direct string comparison and also adapts to sequencing errors. Next, for each pair of reads the most likely matches in the HLA library are determined using Q-values and the predefined tree structure. The third step evaluates the linkage between all matches of each locus and exon and orders them by significance. The fourth step classifies the called alleles as results or discards them as crossover products, statistical noise, co-amplifications etc. For each locus, allele pairs are obtained by combining the respective exon information. Although neXtype reliably rates its results, a user can override the result classification if deemed necessary and declare results as artifacts for future reference. Results NGS results by neXtype have successfully been cross-validated with Sanger sequencing results of 6840 loci. In average, 98.7% of all typings have high resolution. 94.9% do not require user interaction, 2.7% require user editing for improvement of the result, and in 2.4 % data quality was insufficient to allow HLA typing. This high degree of automation reduces analysis time to less than 2 h per 2000 loci. Conclusions NeXtype matches the needs of high throughput HLA labs providing accurate typing with minimal user intervention. Currently, 9,000 loci are typed per day.
