Project: NGS on Reference cell lines

NGS HLA genes typing of Reference Cell Lines and Quality Control

Project leader: Tami Vayntrub and Kazutoyo Osoegawa

Detailed project description:

Cell lines (collected historically by the previous workshops) were sequenced by multiple laboratories to obtain a complete NGS of full-length HLA genes typing. The consensus NGS HLA typing of 406 cell lines, extensively tested by NGS HLA typing techniques during the 17th IHIWS, now serve as unambiguous reference for the evaluation of reagents and platforms currently in use as well as evaluation of new emerging technologies. NGS HLA quality control panels build from these cell lines, will be used for performance validation for NGS HLA typing at the 18th IHIWS. Quality Control panels may also be used for accreditation by the various certification boards.

Additional 364 cell lines, with unknown NGS HLA typing, are available at the Cell Bank and will be offered to participant laboratories to perform NGS HLA typing, with the objective of expanding the reference cell line inventory with NGS HLA typing.

Project participation expectations:

Ordering Information:

  • There are two types of panels that can be ordered:

HLA IHIWS QC Panel (SPD40303):

Each Quality Control Panel consists of DNA from 24 reference cell lines, constructed using  reference cell lines with available consensus NGS HLA typing. Each Laboratory wishing to demonstrate NGS proficiency and participate in NGS HLA Projects, will order ONE panel. Participating laboratory wishing to submit NGS HLA typing data using more than one technology, are expected to demonstrate proficiency in each technology by performing NGS HLA typing using one NGS HLA Quality Control panel per technology. QC panels may also be used for accreditation by the various certification boards.

HLA IHIWS Unknown Panel (SPD40304):

Four different panels available, a total 96 cells. Each Unknown Panel consists of DNA from 24 reference cell lines not tested by NGS but previously typed by multiple techniques such as Sanger sequence Based Typing (SBT), sequence-specific primers (SSP), sequence-specific oligonucleotide probe (SSO) and serological and cellular methods. We encourage participants to order as many as all FOUR panels of 24 cells each. Laboratories who wish to participating in the Unknown panel testing are expected to have demonstrated NGS HLA typing proficiency. NGS HLA typing by multiple laboratories, reagents and platforms will be analyzed, consensus will be reported and published

Instructions to request panels:

    1. Use the Nonprofit Order Form or download the form from the cell bank (rcb@fhcrc.org). For commercial vendors, please use the Corporate order form.
    2. Fill in the requested Information (pink section of the form)
    3. Select the type of panels desired by entering the number of panels in column H of the form (The price is automatically calculated in column K based on the number of panels ($120.00/panel plus a onetime handling fee ($30.31))
    4. You may order QC or/and Unknown panels in the same form
    5. Select Fedex Account desired at the bottom of the form
    6. Completed order forms can be submitted by email attachment to rcb@fhcrc.org or by fax to 206-667-5255
    7. Please copy Tamara at tamara.Vayntrub@Stanford.edu so we may keep track of your participation.
  • Participating laboratory wishing to submit NGS HLA typing data using more than one technology, are expected to demonstrate proficiency in each technology by performing NGS HLA typing using one NGS HLA Quality Control panel per technology (see section below “Results Evaluation and proficiency grading” for proficiency guidelines).
  • Once NGS HLA typing proficiency has been demonstrated, participating laboratories may request to participate in the unknown NGS HLA typing part of the project, and request as many unknown panels of 48 cell each as desired.
  • Participants will request desired number and type of panels via the official IHWG Cell Bank request form. The request form and cost will be determined by the IHWG Cell and Gene Bank.
  • The panels will be constructed of various number of DNA, each labelled with a coded workshop sample ID and shipped directly from the Cell Bank to the requestors with specific instructions for genomic DNA handling and terms and conditions of use.

Typing requirements for quality control panels:

  • All 24 cell lines included in the quality control panel are to be tested by the participating laboratory.
  • Laboratories are expected to perform NGS HLA typing using the platform and reagents of choice.
  • Participants will submit complete results for NGS HLA typing on all 11 loci HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DRB3, HLA-DRB4 and HLA-DRB5.
  • Participants who wish to perform NGS HLA typing for less than 11 loci will need to submit a request in writing to the project leaders for consideration.
  • Drop outs or no typing will be regarded as discrepancies (discordant scoring)
  • When novel alleles are encountered, the closest allele should be reported
  • Special attention should be taken to resolve ambiguities when clinically significant null alleles are detected in the ambiguity string.

Typing requirements for unknown panels:

  • Participants must have demonstrated proficiency in NGS HLA typing (see “Results Evaluation and proficiency grading” below)
  • Participants may choose the number of unknown panels (48 DNA in each) to type.
  • Laboratories are expected to perform NGS HLA typing using the platform and reagents of choice.
  • Participants will submit complete results for NGS HLA typing on all 11 loci HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DRB3, HLA-DRB4 and HLA-DRB5.
  • Participants who wish to perform NGS HLA typing for less than 11 loci will need to submit a request in writing to the project leaders for consideration.
  • Only data deemed proficient will be included in the post workshop publications and consensus analysis.

Data Submission:

  • The results of the NGS HLA gene analysis as well as the raw data (example: fastq files) will be uploaded to the 18th IHIW database
  • NGS HLA genotypes will be uploaded in Genotype List (GL) String format
  • Other associated meta-data such as consensus sequences, the reagents, sequencing instrument, software and information about the IPD-IMGT/HLA database version will also be imported into the 18th IHIWS database.
  • Specific instructions to upload data to the 18th IHIWS will be communicated to the participants upon enrolment. The method to submit and store the raw data will be communicated soon after.
  • To participate in the analysis and follow up authorship in the post workshop publications, NGS HLA typing and associated metadata is expected to be uploaded by each of the projects defined deadline.

Results Evaluation and proficiency grading:

  • Proficiency requires a minimum of 95% concordance at the HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DRB3, HLA-DRB4 and HLA-DRB5 for NGS HLA projects where participants are expected to submit data for all 11 loci. For projects where only HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1 will be analysed or considered, participants may request an exception when submitting results of the quality control panel requested.
  • Evaluation of the results submitted for the quality control panel, will be performed using the HLAGenotypeEvaluator software (https://github.com/IHIW/hlaGenotypeEvaluator). Each allele of the 11 HLA loci for each cell line, will be scored against the know NGS HLA typing of the cell line performed during the 17th
  • Failure to submit a typing for any of the DNA in the quality control panel, will be scored as discordant for that cell.
  • The results of the evaluation will be reported to the participant once analysis is completed. Project leaders may request clarification and participant may submit a subsequent typing if ambiguity resolution was obtained at a later date. Provided that data is submitted prior to the given deadline.
  • Scoring will be performed as reported in the 17th workshop publication “Quality control project of NGS HLA genotyping for the 17th International HLA and Immunogenetics Workshop”.
  • Scoring definition and examples:
    ScoreCompared to Reference Cell Line typingSubmitted type 17th IHIWS NGS typing (reference cell line)Overall analysis
    IdenticalGenotypes are exactly identicalA*68:01:02:01 A*68:01:02:01Identical or concordant
    ConcordantConcordant at least by two field assessmentDPA1*01:03:01:01DPA1*01:03:01:02
    AmbResult
    Concordant
    Ambiguity in the submitted type, not reported in the referenceDQB1*05:03:01:01/
    DQB1*05:03:01:02
    DQB1*05:03:01:01Ambiguous
    AmbRef
    Concordant
    Ambiguity reported in the reference but not in the submitted typeDQA1*05:05:01:05DQA1*05:05:01:01/
    DQA1*05:05:01:02
    AmbRefAmb
    Result
    Concordant
    Ambiguity in both, the reference and submitted typeDRB1*07:01:01:02/
    DRB1*07:01:01:01/
    DRB1*07:01:01:03
    DRB1*07:01:01:01/
    DRB1*07:01:01:02
    UnresolvedNull
    AmbResult
    Concordant
    Concordant, but null allele was found in the submitted type ambiguity stringB*15:01:01:01/
    B*15:01:14/
    B*15:26N
    B*15:26NUnresolved Null
    DiscordantIndicates both alleles do not match by two field assessmentB*57:12B*57:02:01Discordant
  • A score of “Not Reported” will only be considered for the exceptional situation where not all loci NGS HLA typing is submitted with prior approval by the project leaders.

Milestones in years:

2019:

  1. Submit a complete NGS HLA typing for 406 cell from consensus analysis from 17th IHIWS projects and subsequent publications to the Fred Hutchinson Cancer Research Cell Bank.
  2. Identify available cell lines with complete NGS typing and cell lines with unknown NGS typing.
  3. Grow selected cell lines
  4. Creation of cell line panels. Two kind of panels will be made available: (1) Quality control panels with consensus NGS typing performed during the 17th IHIWS (total of 406 cell lines available) and (2) Unknown cell line panels, consisting of 362 cell lines pending NGS typing. This will be done in coordination with Fred Hutchinson Cancer Research Cell Bank. The number of panels to be created will need to be defined, based on a calculation on the number of participating laboratories enrolling in the project.
  5. Order Forms for panel requests to be made available to all participants of the project. Pricing and instructions to be finalize.
  6. Finalize timelines for data submission, analysis and participant feedback
  7. Notification to the community via News Letter regarding the goals of the project and logistics/instructions to order.
  8. In coordination with the database owners, identify data infrastructure required:
    • IPD-IMGT/HLA Database version reference to fix analysis
    • Describe specific instructions to upload data for this project to the 18th IHIWS
    • Identify mechanism to submit and store fastq or other raw data files to the Workshop database
    • Define the expectation for typing and data submission for upload in the database
    • Number of minimum loci to test and report for each, quality control and additional reference cells NGS typing
    • Minimum number of cell required to type for proficiency program
    • Definition of concordance, discrepancy, ambiguity and maximum discordance expectation to qualify as proficient. (null alleles) discordant includes no typing
    • Define the expectation for typing and data submission for upload in the database

2020:

Upload NGS typing into the database. Complete analysis of quality control data and provide participants with feedback on results. Qualification of proficiency for participation in other NGS projects data submission, including Unknown panel participation.

2021:

Final analysis of reference cell lines NGS typing consensus and quality control panels results


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