Research ArticleImmunology and Cancer

Measurement and Clinical Monitoring of Human Lymphocyte Clonality by Massively Parallel V-D-J Pyrosequencing

See allHide authors and affiliations

Science Translational Medicine  23 Dec 2009:
Vol. 1, Issue 12, pp. 12ra23
DOI: 10.1126/scitranslmed.3000540
  • Fig. 1

    Bar-coded PCR amplicons for multiplexed IgH sequencing. PCR primers used for preparing bar-coded amplicons for high-throughput sequencing were designed with the FR2 IgH V gene segment family primers and the common IgH J segment primer from the BIOMED-2 consortium (19). Additional sequences required for emulsion PCR and pyrosequencing were added (indicated in green) at the 5′ end of the IgH-specific primers. In addition, a 6-, 7-, or 10-nucleotide sequence bar code was designed into the modified IgH J primer to identify the sample from which the PCR amplicons were derived. In the specimens analyzed with the 454 Titanium sequencer, an additional 10-nucleotide sample bar code was incorporated into the multiplexed IgH V gene segment primers used for amplification (table S1). Lines with arrowheads indicate PCR primers. Green segments, primer sequences needed for 454 sequencing protocol; red segments, V gene segment sequence; gray segments, nontemplated N base sequences; yellow segments, D gene segment sequence; blue segments, J gene segment sequence; green ellipse, sample-specific bar code enabling pooling of IgH libraries for multiplexed sequencing. Samples 1 and 2 could represent DNA template from any two clinical specimens or independent DNA template aliquots from the same specimen.

  • Fig. 2

    IgH V and J gene segment usage in healthy peripheral blood, oligoclonal or indeterminate specimens, and lymphoid malignancy specimens. Bar-coded IgH rearrangement libraries were PCR amplified from genomic DNA of human specimens, pooled, and characterized by high-throughput pyrosequencing. Experiments 1 and 2 were independent experimental replicates beginning with different aliquots of the template DNA from each specimen. Each wide row represents the IgH sequences identified in a single sample. Samples (S1 to S19) are labeled at the far left. The x axis (across the top of the panels) indicates the V gene segment used in the receptor, and the y axis (the column at the left of the panels) within each wide row represents the J gene segments used. The size and color of the circle at a given point indicates what proportion of all sequences in the sample used that particular combination of V and J gene segments. Sequences in which V, D, or J segments or junctions could not be unambiguously assigned were filtered before generation of these plots. rep, replicate sequence pool PCR amplified from an independent aliquot of template DNA; CLL, chronic lymphocytic leukemia; FL, follicular lymphoma; SLL, small lymphocytic lymphoma; PTLD, posttransplant lymphoproliferative disorder; dil, dilution.

  • Fig. 3

    Titration of a chronic lymphocytic leukemia clonal sample into healthy peripheral blood. Pooled bar-coded IgH library sequencing was carried out on a series of 10-fold dilutions of a chronic lymphocytic leukemia blood sample (sample 13) into a healthy control blood sample (sample 14) to evaluate the sensitivity and linearity of high-throughput sequencing for detection of a known clonal sequence. The percentage of sequences matching the chronic lymphocytic leukemia clone in each diluted specimen is plotted on a log scale, with zero indicating that no sequences were detected. The counts of clonal sequences in each sample were as follows: CLL sample, 7805 clonal of 8612 total; healthy blood control, 0 clonal of 7518 total; 1:10 dilution, 2095 clonal of 13,717 total; 1:100 dilution, 156 clonal of 8674 total; 1:1000 dilution, 23 clonal of 9471 total; 1:10,000 dilution, 3 clonal of 8895 total; 1:100,000 dilution, 0 clonal of 6940 total. The negative control is the healthy donor blood sample used for diluting the clonal CLL sample. A second experiment measuring fewer sequences from independent PCR amplifications from the same samples detected the following number of clonal sequences in each sample: CLL sample, 422 clonal of 566 total; healthy blood control, 0 clonal of 270 total; 1:10 dilution, 189 clonal of 665 total; 1:100 dilution, 11 clonal of 230 total; 1:1000 dilution, 0 clonal of 344 total; 1:10,000 dilution, 0 clonal of 329 total; 1:100,000 dilution, 0 clonal of 208 total.

  • Table 1

    Patient specimens for IgH sequencing. The clonality assay results are those obtained with standard PCR amplification and capillary electrophoresis of product amplicons. Blood, peripheral blood mononuclear cells; Lymph node, formalin-fixed, paraffin-embedded lymph node tissue; Liver, formalin-fixed, paraffin-embedded liver tissue; CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; FL, follicular lymphoma; PTLD, posttransplant lymphoproliferative disease; DLBCL, diffuse large B cell lymphoma.

    No.DescriptionSample typeClonality assay result
    1Healthy donor 1, time 0BloodNegative
    2Healthy donor 1, time 0BloodNegative
    3Healthy donor 1, time 14 monthsBloodNegative
    4Healthy donor 1, time 14 monthsBloodNegative
    5Patient 1; CLL/SLL time 0BloodPositive
    6Patient 1; CLL/SLL time 3 monthsBloodPositive
    7Patient 2; FLLymph nodePositive
    8Patient 3; FL and SLL in lymph nodeLymph nodePositive
    9Patient 4; CLL/SLLBloodOligoclonal
    10Patient 5; PTLD, marrow infiltrateBone marrowPositive
    11Patient 5; PTLD, liver DLBCLLiverPositive
    12Healthy donor 2BloodNegative
    13Patient 6; CLLBloodPositive
    14Healthy donor 3BloodNegative
    15Patient 6 CLL diluted 1:10BloodPositive
    16Patient 6 CLL diluted 1:100BloodNegative
    17Patient 6 CLL diluted 1:1000BloodNegative
    18Patient 6 CLL diluted 1:10,000BloodNegative
    19Patient 6 CLL diluted 1:100,000BloodNegative
  • Table 2

    Comparison of high-throughput sequencing with real-time PCR MRD monitoring assays. For each patient specimen, IgH rearrangements were amplified from 200 ng of genomic DNA of the indicated specimen types with bar-coded primers adapted for 454 pyrosequencing. The IgH rearrangement libraries were pooled and sequenced. The number of clonal sequences (matching the initial diagnostic specimen clone) and the total number of sequences obtained are listed. Data from pyrosequencing were compared to the results of custom quantitative real-time PCR assays designed to amplify the patient’s malignant clonal sequence. The RT-PCR results were considered positive if >100 copies per microgram of template DNA were detected.

    PatientSpecimenClone copies*Total sequences%Clone copiesTotal sequences%RT-PCR (copies/μg)
    CLL A sample 1Diagnostic lymph node7,22711,19064.65,7458,93564.3>100,000
    CLL A sample 2Blood03410.006700.010
    CLL A sample 3Blood381,4772.6603,3501.81,485
    CLL A sample 4Blood05880.001,6570.091
    CLL A sample 5Blood04300.004910.037
    CLL A sample 6Bone marrow01,4710.0212,9910.7314
    CLL B sample 1Diagnostic bone marrow2,4614,36356.41,9643,58154.8>100,000
    CLL B sample 2Bone marrow1,0801,97454.71,6563,00255.25,496
    CLL B sample 3Blood01620.002080.024
    CLL B sample 4Blood01140.001170.010
    CLL B sample 5Bone marrow18849338.13431,12730.4944
    Unrelated CLLBlood05,3260.007,6730.0
    Normal controlTonsil014,0070.005,1670.0

    *First replicate.

    Second replicate.

    • Table 3

      Coincident sequences in a healthy donor’s peripheral blood at two time points. IgH rearrangements from peripheral blood mononuclear cells of a healthy blood donor were PCR amplified in multiple independent replicate PCR reactions and sequenced. The table shows the number of identical sequences detected in more than one replicate (termed coincident sequences). Blood samples from two time points separated by 14 months were analyzed. Sequences from different replicates were considered to be coincident sequences if they shared the same V, D, and J segment usage as well as the same V-D and D-J junctional nucleotide sequences. T1, initial time point; T2, second time point 14 months later; r1 through r7, replicates 1 through 7.

      T1r2T1r3T1r4T1r5T1r6T2r1T2r2T2r3T2r4T2r5T2r6T2r7
      T1r1221112120221
      T1r211043001010
      T1r30200010010
      T1r4120011031
      T1r511001022
      T1r60000021
      T2r1011111
      T2r202201
      T2r31202
      T2r4202
      T2r510
      T2r65
    • Table 4

      Coincident IgH sequences in peripheral blood of healthy donors of various ages. Peripheral blood samples from 23 healthy donors of ages ranging from 19 to 79 years were analyzed by deep sequencing IgH rearrangements in six replicates from each sample. The number of distinct sequences detected in more than one replicate (termed coincident sequences) from each individual is tabulated below. Sequences from different replicates were considered to be coincident sequences if they shared the same V, D, and J segment usage as well as the same V-D and D-J junctional nucleotide sequences. Calculation of the minimum IgH repertoire diversity in each patient, as indicated by the number of coincident sequences detected, is described in the Supplementary Material.

      AgeTotal sequencesCoincidencesMinimum diversity
      23456
      1919,3682200002,136,616
      2012,598610200704,883
      236,9641100001,133,759
      256,5221000001,328,380
      314,086101100474,366
      326,112900001,328,380
      355,358400001,860,053
      375,253411001,973,903
      382,17318210070,876
      424,094110000381,515
      442,24930000438,241
      456,781652200325,619
      457,6971200001,409,687
      506,841610001,718,401
      5410,8221310013,369,228
      553,42670000513,469
      605,17383000704,883
      615,092100006,349,446
      687,0281112011,897,254
      705,5521000001,276,797
      757,064501003,303,164
      785,895400003,051,613
      797,1271100001,587,537

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/1/12/12ra23/DC1

      Materials and Methods

      Fig. S1. V-D-J plots of healthy peripheral blood and lymphoid malignancies.

      Fig. S2. Sequence complexity of healthy donor blood specimens.

      Table S1. Sequencing primers.

      Table S2. Patient specimens used in experiment 2.

      Table S3. Number of sequences determined per specimen.

      Table S4. Sequences found in more than one replicate from healthy donor blood samples.

      References

    • Supplementary Material for:

      Measurement and Clinical Monitoring of Human Lymphocyte Clonality by Massively Parallel VDJ Pyrosequencing

      Scott D. Boyd, Eleanor L. Marshall, Jason D. Merker, Jay M. Maniar, Lyndon N. Zhang, Bita Sahaf, Carol D. Jones, Birgitte B. Simen, Bozena Hanczaruk, Khoa D. Nguyen, Kari C. Nadeau, Michael Egholm, David B. Miklos, James L. Zehnder, Andrew Z. Fire*

      *To whom correspondence should be addressed. E-mail: afire{at}stanford.edu

      Published 23 December 2009, Sci. Transl. Med. 1, 12ra23 (2009)
      DOI: 10.1126/scitranslmed.3000540

      This PDF file includes:

      • Materials and Methods
      • Fig. S1. V-D-J plots of healthy peripheral blood and lymphoid malignancies.
      • Fig. S2. Sequence complexity of healthy donor blood specimens.
      • Table S1. Sequencing primers.
      • Table S2. Patient specimens used in experiment 2.
      • Table S3. Number of sequences determined per specimen.
      • Table S4. Sequences found in more than one replicate from healthy donor blood samples.
      • References

      [Download PDF]

    Stay Connected to Science Translational Medicine

    Navigate This Article