scholarly journals High-throughput sequencing of murine immunoglobulin heavy chain repertoires using single side unique molecular identifiers on an Ion Torrent PGM

Oncotarget ◽  
2018 ◽  
Vol 9 (54) ◽  
pp. 30225-30239 ◽  
Author(s):  
Jean-Philippe Bürckert ◽  
William J. Faison ◽  
Danielle E. Mustin ◽  
Axel R.S.X. Dubois ◽  
Regina Sinner ◽  
...  
Keyword(s):  
High Throughput ◽  
Heavy Chain ◽  
High Throughput Sequencing ◽  
Immunoglobulin Heavy Chain ◽  
Ion Torrent ◽  
Ion Torrent Pgm ◽  
Single Side

scholarly journals High-throughput sequencing of murine immunoglobulin heavy chain transcripts using single side unique molecular identifiers on an Ion Torrent PGM

2017 ◽  
Author(s):  
Jean-Philippe Bürckert ◽  
William J. Faison ◽  
Axel R. S. X. Dubois ◽  
Regina Sinner ◽  
Oliver Hunewald ◽  
...  
Keyword(s):  
High Throughput ◽  
Heavy Chain ◽  
High Throughput Sequencing ◽  
Gene Segment ◽  
Personal Genome ◽  
Sequencing Platform ◽  
Ion Torrent Pgm ◽  
Indel Detection ◽  
Processing Strategies ◽  
Nucleotide Insertions

AbstractWith the advent of high-throughput sequencing (HTS), profiling immunoglobulin (IG) repertoires has become an essential part of immunological research. Advances in sequencing technology enable the IonTorrent Personal Genome Machine (PGM) to cover the full-length of IG mRNA transcripts. Nucleotide insertions and deletions (indels) are the dominant errors of the PGM sequencing platform and can critically influence IG repertoire assessments. Here, we present a PGM-tailored IG repertoire sequencing approach combining error correction through unique molecular identifier (UID) barcoding and indel detection through ImMunoGeneTics (IMGT), the most commonly used sequence alignment database for IG sequences. Using artificially falsified sequences for benchmarking, we found that IMGT efficiently detects 98% of the introduced indels through gene-segment frameshifts. Undetected indels are either located at the ends of the sequences or produce masked frameshifts with an insertion and deletion in close proximity. IMGT’s indel correction algorithm resolves up to 87% of the tested insertions, but no deletions. The complementary determining regions 3 (CDR3s) are returned 100% correct for up to 3 insertions or 3 deletions through conservative culling. We further show, that our PGM-tailored unique molecular identifiers results in highly accurate HTS datasets if combined with the presented data processing. In this regard, considering sequences with at least two copies from datasets with UID families of minimum 3 reads result in correct sequences with over 99% confidence. The protocol and sample processing strategies described in this study will help to establish benchtop-scale sequencing of IG heavy chain transcripts in the field of IG repertoire research.

scholarly journals Next Generation Amplicon Sequencing of Immunoglobulin Heavy Chain Gene Rearrangaments for Minimal Residual Disease (MRD) Stratification in Childhood Acute Lymphoblastic Leukemia (ALL): A Comparison with Classical qPCR-Based Technique

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2395-2395
Author(s):  
Michaela Kotrova ◽  
Katerina Muzikova ◽  
Ester Mejstrikova ◽  
Michaela Novakova ◽  
Violeta Bakardjieva-Mihaylova ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Risk Group ◽  
Immunoglobulin Heavy Chain ◽  
Patient Specific ◽  
Ion Torrent ◽  
Next Generation ◽  
Childhood All ◽  
Ion Torrent Pgm ◽  
Ion Proton ◽  
Group Stratification

Abstract Introduction MRD is an important predictor of outcome in childhood ALL. Since 2000, MRD detected by quantitative PCR (qPCR) for immunoglobulin and T-cell receptor gene rearrangements with a minimal sensitivity of 1E-04 has been used for risk group stratification in pediatric BFM trials. Next generation sequencing (NGS) permits rapid parallel sequencing of large numbers of DNA segments. It can overcome most of the limitations of qPCR: it allows highly specific molecular detection of MRD without laborious optimization of patient-specific assays. Moreover it enables not only monitoring of malignant clone but also shows the picture of entire immune background. Aims To develop an assay for immunoglobulin heavy chain (IgH) rearrangements detection on Ion Torrent PGM/Ion Proton platforms and compare the MRD levels with qPCR at BFM stratification timepoints. Methods Two round PCR was used for library preparation. Libraries were created from 450ng (equivalent of 70,000 DNA copies) of bone marrow DNA and 50ng of Human Genomic DNA (Roche). In the first round of PCR rearranged IgH genes were amplified using IGH FR3 BIOMED-2 primers (van Dongen, Leukemia 2003). In the second round the sequencing adapters and multiplex identifiers were added. Sequencing was performed on Ion Torrent PGM/Ion Proton sequencers using a 200bp chemistry. We developed a bioinformatics algorithm for detection of reads with known clonal V-D-J rearrangements within the resulting fastq files. For validation of the assay we sequenced 1E-1 to 1E-5 dilutions of diagnostic samples from 2 patients in multiplicates. The results show that the assay gives reproducible quantitative results up to 1E-4 dilution. Results We sequenced 183 samples from 67 patients (52×day 15, 65×day 33, 66×day 78) with childhood ALL treated according to AIEOP-BFM ALL 2000 protocol with the median coverage 587,406 reads per sample. Eighty-three (45.4%) samples were negative by both methods. Fifteen (8.2%) samples were positive by NGS and negative by qPCR and 14 (7.7%) samples were positive by qPCR and negative by NGS. All the discordant samples had MRD levels below the sensitivity of both methods. The overall correlation of all double positive and double negative samples was very good (R2=0.93). If risk group stratification based on NGS results would be performed, 8 patients would be classified as intermediate risk (IR) instead of standard risk (SR) (one of whom relapsed) and 8 patients as SR instead of IR. One patient would be relocated from IR to slow early responders (SER) group, and two patients from SER to IR (one of them relapsed). One patient who relapsed would be classified as high risk (HR) instead of SER. All 5 patients who were MRD negative at d15 by NGS remained MRD negative in later timepoints and none of them relapsed. Discussion We present a cost-effective and widely adoptable NGS-based method that provides clinically relevant results in childhood ALL. NGS has a great potential to reduce the laboriousness associated with patient-specific qPCR analysis and to speed up the process of MRD detection. The sensitivity of both methods is comparable when ~500ng of DNA is used. The majority of the differences were in the samples with MRD levels below 1E-4 and most of treatment stratification changes occurred between SR/IR. However, the different stratification mostly concerned patients who did not relapse. The sensitivity of NGS could be improved if more DNA was analyzed. However, the benefit of increased MRD sensitivity is questionable due to possible overtreatment of patients with very low MRD loads after induction treatment. “Online” identification of d15 MRD negative patients reported as having an excellent prognosis previously is possible only by NGS, because optimization of patient-specific qPCR takes several weeks. The addition of 10% polyclonal DNA seems to solve the problem of MRD overestimation by NGS in samples with B-cell aplasia. At present, the main drawback of the Ig/TCR-exploring NGS methods is lack of standardization both in the experimental setting and in data analysis. Therefore, recently a European network, the EuroClonality NGS Consortium, has been formed to standardize the whole workflow of analytics, pre-analytics and bioinformatics not only for MRD quantification but also for clonality assessment in lymphoid neoplasms and for repertoire analysis. Supported by IGA NT14343, IGA NT12397, IGA NT13462-4 and GAUK 394214. Disclosures No relevant conflicts of interest to declare.

scholarly journals Towards the validation of high-throughput sequencing (HTS) for routine plant virus diagnostics: measurement of variation linked to HTS detection of citrus viruses and viroids

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Rachelle Bester ◽  
Glynnis Cook ◽  
Johannes H. J. Breytenbach ◽  
Chanel Steyn ◽  
Rochelle De Bruyn ◽  
...  
Keyword(s):  
High Throughput ◽  
Extraction Method ◽  
Pathogen Detection ◽  
High Throughput Sequencing ◽  
Expression Profiles ◽  
Rna Extraction ◽  
Healthy Plant ◽  
Ion Torrent ◽  
Extraction Protocol ◽  
Zymo Research

Abstract Background High-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The validation of HTS-based pathogen detection is therefore paramount. Methods Plant infections were established by graft inoculating a suite of viruses and viroids from established sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA extraction method, platform used and bioinformatic analysis. Results The study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison between samples across different protocols. Conclusions This study highlights the need to measure the level of variation that can arise from the different variables of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously used, but with the necessary validations and standard operating procedures, the implementation of HTS as part of routine pathogen screening practices is possible.

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