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Targeting the JAK/STAT Pathway in Cytotoxic T lymphocytes (CTL) by Next Generation Sequencing (NGS)

  • Maddalena GiganteEmail author
  • Sterpeta Diella
  • Elena Ranieri
Protocol
  • 2.1k Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 1186)

Abstract

Next Generation Sequencing (NGS), together with our evolving knowledge of genes and disease, is likely to change the current practice of medicine and public health by facilitating more accurate, sophisticated, and cost-effective genetic testing. Here, we propose a new molecular approach by using MiSeq Sequencing System (Illumina) to investigate the presence of mutations/variants in genes of JAK/STAT pathway involved in different cytotoxic T lymphocytes (CTL)-mediated immune disorders and to develop and validate new and less expensive molecular protocol based on Next Generation Sequencing.

Key words

Mutation NGS Cytotoxic T lymphocytes 

1 Introduction

Next Generation Sequencing (NGS), together with our evolving knowledge of genes and disease, is likely to change the current practice of medicine and public health by facilitating more accurate, sophisticated, and cost-effective genetic testing. Here, we propose a new molecular approach by using MiSeq Sequencing System (Illumina). Primary objectives of the present methodology will be to investigate the presence of genetic abnormalities in genes of JAK/STAT pathway involved in different cytotoxic T lymphocytes (CTL)-mediated immune disorders and to develop and validate new and less expensive molecular protocol based on Next Generation Sequencing. Cytotoxic T cells or natural killer cells have played important roles in the setting of infectious disease and cancer. Though many immune components that participate in these processes are known, the underlying mechanisms remain poorly defined. Cytokines and their receptors play an essential role in T cell proliferation and activation. Although cytokine receptors lack intrinsic kinase activity, they are associated with cytoplasmic protein tyrosine kinases (PTKs) that phosphorylate downstream signaling molecules such as the signal transducers and activators of transcription (STATs). Activated STATs, in turn, translocate to the nucleus and regulate gene expression [1]. The Janus kinase (JAK) family of non-receptor PTKs includes critical elements in cytokine signaling. To date, four JAKs (JAK1, JAK2, JAK3, TYK2) and seven STATs (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6) have been described and fully characterized [2]. Mutations in JAK3 gene have been associated with autosomal recessive severe combined immunodeficiency (SCID), a condition in which T cell development and B cell function are severely reduced, indicating that this kinase is essential for the correct development of mature lymphoid lineages [3, 4]. Moreover, the association of constitutive activation of JAK/STAT pathway with proliferation of T cell leukemia/lymphoma cells [5] and the presence of activating mutations in the JAK3 pseudokinase domain in the acute megakaryoblastic leukemia (AMKL) cell line [6] suggest a role for altered JAK3 expression and/or function in the development of human cancer. An association between JAK/STAT pathway and Renal Cell Carcinoma (RCC), a highly immunogenic tumor, was demonstrated by our group [7, 8] and by Kolenko et al. [9]. Finally, activating mutations in STAT3 were recently identified in 40 % of patients with large granular lymphocytic leukemia, a disorder characterized by the presence of abnormal CD3+CD8+CD57+ lymphocytes corresponding to activated effector cytotoxic T lymphocytes (CTLs) [10]. Our capability to couple specific phenotypes to genetic variation is now unprecedented, as high-resolution genome wide approaches are able to uncover novel relationships and rare variants. Next Generation Sequencing (NGS) has the advantage to harvest all the genetic variations, both small variants (single base substitution, small INDELs) and structural variants, and to identify also rare variants in genes belonging to a functionally relevant pathway and the whole spectrum of variability associated with a specific clinical/phenotypic spectrum. Since serially testing each gene of pathway by Sanger sequencing would be time consuming and expensive, we propose a multi-gene protocol based on NGS for a panel of 11 selected genes (JAK1, JAK2, JAK3, TYK2, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6) already known to be involved in JAK/STAT pathway and immune mechanisms. The development and validation of a protocol based on NGS may act as a model for tracing the new way of genomic laboratory medicine and have a strong potential also for clinical and diagnostic applications. Considering the key role of JAKs/STATs pathway in immune mechanisms, here, we set up a protocol using Next Generation Sequencing (NGS) for targeted analysis of JAK/STAT pathway genes and validate it by comparing the results with standard Sanger method. A training set of samples was used to optimize the entire process, and a second set was used to validate and independently evaluate the performance of the workflow. By this validation we are able to evaluate if our NGS protocol is able to ensure appropriateness, with high sensitivity and specificity, if it is cheaper, faster, and with a higher reproducibility than the Sanger reference method. Although in the first instance our protocol has a major impact on the studied genes, the implications may extend beyond, acting as a model for the new course of genomic medicine.

2 Materials

  1. 1.

    DNA purification kit (Life Technologies, Qiagen, Hilden, Germany).

     
  2. 2.
     
  3. 3.
     
  4. 4.

    Human JAKs/STATs gene flanking intronic forward and reverse primers.

     
  5. 5.

    TaqGold (Life Technologies).

     
  6. 6.

    PCR Thermocycler.

     
  7. 7.

    Big Dye Terminator v3.1 cycle sequencing kit (Life Technologies).

     
  8. 8.

    3130 Genetic Analyzer (Life Technologies).

     
  9. 9.

    SeqScape program (Life Technologies).

     
  10. 10.

    Design Studio (Illumina) software.

     
  11. 11.

    TruSeq Custom Amplicon Library Preparation Kit (Illumina).

     
  12. 12.

    TruSeq Custom Amplicon Library Preparation Guide (CATALOG: FC-130-9005DOC).

     
  13. 13.

    ACD1 and ACP1: Amplicon Control DNA 1 and Amplicon Control Oligo Pool (Illumina).

     
  14. 14.

    OHS2: Oligo Hybridization for Sequencing Reagent 2 (Illumina).

     
  15. 15.

    ELM4: Extension Ligation Mix 4 (Illumina).

     
  16. 16.

    PMM2: PCR Master Mix 2 (Illumina).

     
  17. 17.

    TDP1: TruSeq DNA Polymerase (Illumina).

     
  18. 18.

    SW1: Stringent Wash 1 (Illumina).

     
  19. 19.

    UB1: Universal Buffer 1 (Illumina).

     
  20. 20.

    LNB1 Library Normalization Beads 1 (Illumina).

     
  21. 21.

    HT1 Hybridization Buffer (Illumina).

     
  22. 22.

    LNA1 Library Normalization Additives 1 (Illumina).

     
  23. 23.

    LNW1 Library Normalization Wash 1 (Illumina).

     
  24. 24.

    LNS2 Library Normalization Storage Buffer 2 (Illumina).

     
  25. 25.

    EBT Elution Buffer with Tris (Illumina).

     
  26. 26.

    TruSeq Custom Amplicon Library Preparation Index Kit (Illumina).

     
  27. 27.

    CAT Custom Amplicon oligo Tube (Illumina).

     
  28. 28.

    i5 Index Primers, A501–A508 (Illumina).

     
  29. 29.

    i7 Index Primers, A701–A712 (Illumina).

     
  30. 30.

    10 N NaOH.

     
  31. 31.

    96-well skirted PCR plates, 0.2 ml, polypropylene.

     
  32. 32.

    Eppendorf microcentrifuge tubes (screw top recommended).

     
  33. 33.

    Conical tubes, 15 ml.

     
  34. 34.

    Adhesive aluminum foil seal.

     
  35. 35.

    Microseal “A” adhesive seals.

     
  36. 36.

    PCR Eight-Tube Strips.

     
  37. 37.

    Agarose gel.

     
  38. 38.

    Agilent 2100 Bioanalyzer.

     
  39. 39.

    DNA molecular weight markers.

     
  40. 40.

    MiSeq Sequencing System (Illumina).

     
  41. 41.

    Filter plate with lid (Iluumina).

     
  42. 42.

    Adapter collar (Illumina).

     
  43. 43.

    MIDI plates.

     
  44. 44.

    AMPure XP Beads (Beckman Coulter).

     
  45. 45.

    96 well magnet plate (Beckman Coulter).

     

3 Methods

3.1 Human DNA Isolation and Sample Selection

The collection and handling of human blood should be performed in accordance to institutional rules and guidelines. Assume all blood products contain infectious material.
  1. 1.

    Isolate Genomic DNA from human peripheral blood leukocytes using standard DNA Purification Kit according to manufacturing’ instructions (Life Technologies, Qiagen).

     
  2. 2.

    Measure DNA concentration and purity with NanoDrop and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), providing highly accurate concentration measures of extremely small samples and sensitive quality analysis of DNA samples.

     
  3. 3.

    A training set of DNA samples (15–20), previously fully Sanger sequenced for selected genes of JAK/STAT pathway, should be used to optimize the entire process and a second set (10–15) should be used to validate and independently evaluate the performance of the workflow.

     
  4. 4.

    The use of samples that were previously fully Sanger sequenced allows the development of a set of known variants to be used as surrogate markers. This set should include known SNPs and different types of mutations (missense, nonsense, and indels) to reflect the range of variants expected to be identified by NGS.

     
  5. 5.

    To analyze the performance of the developed variant prioritization pipeline (VPP), a set of 10–15 additional samples, for which no molecular analysis has been performed before, should be selected.

     

3.2 Design Custom Oligo Probes

  1. 1.

    Use DesignStudio software (Illumina), an easy-to-use online software tool, to design oligo probes of targeting genomic regions of interest.

     
  2. 2.

    Initiate a project by entering target regions of the genome into DesignStudio software. Perform probe design automatically using an algorithm that considers a range of factors, including GC content, specificity, probe interaction, and coverage.

     
  3. 3.

    Candidate amplicons should be visualized and assessed using estimated success scores. Probes could be filtered with user defined tags, and then added to, or removed from the design project.

     
  4. 4.

    Optimize the assay in-house, with additional primers designed to target regions that were not well captured.

     
  5. 5.
    The final assay includes 385 amplicons targeted the protein-coding sequence of 11 selected genes (JAK1, JAK2, JAK3, TYK2, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6) involved in JAK/STAT pathway with an overhang at exon boundaries to capture splice site variants (Table 1).
    Table 1

    Characteristics of 11 genes included in the assay

    Gene symbol

    Gene product

    Genomic locus

    Reference sequence

    Number of exons

    Coding cDNA ?length (bp)

    Number of amplicons

    Disease association

    JAK1

    Tyrosine-protein Kinase JAK1

    1p32.3–p31.3

    NM_002227.2

    25

    3,465 bp

    35

    Variants associated with different type of tumors

    JAK2

    Tyrosine-protein Kinase JAK2

    9p24

    NM_004972.3

    25

    3,399 bp

    36

    Leukemia acute myeloid (AML)

    Myelofibrosis

    Polycythemia Vera

    Thrombocythemia 3

    JAK3

    Tyrosine-protein Kinase JAK3

    19p13.1

    NM_000215.3

    24

    3,375 bp

    37

    Severe combined immunodeficiency T− B+

    Renal Cell Carcinoma

    Leukemia

    Lymphoma

    TYK2

    Non-receptor tyrosine-protein kinase TYK2

    19p13.2

    NM_003331.4

    25

    3,564 bp

    34

    Autosomal recessive hyper IgE syndrome due to TYK2 deficiency

    Juvenile rheumatoid factor-negative polyarthritis

    Oligoarticular juvenile arthritis

    STAT1

    Signal transducer and activator of transcription 1

    2q32.2

    NM_007315.3

    25

    2,253 bp

    36

    Mendelian susceptibility to mycobacterial diseases due to partial STAT1 deficiency

    Candidiasis, Familial Chronic Mucocutaneous, Autosomal Dominant

    STAT2

    Signal transducer and activator of transcription 2

    12q13.3

    NM_005419.3

    24

    2,556 bp

    37

    Variants associated with different type of tumors

    STAT3

    Signal transducer and activator of transcription 3

    17q21.31

    NM_003150.3

    24

    2,310 bp

    44

    Autosomal dominant hyper IgE syndrome

    STAT4

    Signal transducer and activator of transcription 4

    2q32.2-q32.3

    NM_001243835.1

    24

    2,247 bp

    27

    Behcet disease

    Juvenile rheumatoid factor-negative polyarthritis

    Oligoarticular juvenile arthritis

    STAT5A

    Signal transducer and activator of transcription 5A

    17q11.2

    NM_003152.3

    20

    2,385 bp

    30

    Variants associated with different type of tumors

    STAT5B

    Signal transducer and activator of transcription 5B

    17q11.2

    NM_012448.3

    19

    2,364 bp

    34

    Laron syndrome with immunodeficiency

    Growth hormone insensitivity with immunodeficiency

    STAT6

    Signal transducer and activator of transcription 6

    12q13

    NM_001178078.1

    22

    2,544 bp

    35

    Variants associated with different type of tumors

    Total

        

    3,0462 bp

    385

     
     
  6. 6.

    After visualization and Quality Control (QC) oligonucleotide probes are synthesized and pooled into a tube (Custom Amplicon Tube, CAT) containing all the probes necessary to generate the attempted amplicons per reaction.

     
  7. 7.

    Sample-specific indices will be then added to each library by PCR using common primers from TruSeq Amplicon index Kit (see Subheading 3.3).

     

3.3 Amplicon Library Preparation Workflow

  1. 1.
    Prepare your libraries using TruSeq Custom Amplicon Library Preparation Kit and TruSeq Custom Amplicon Library Preparation Index Kit (Illumina) and the protocol detailed in the user guide (Illumina, CATALOG: FC-130-9005DOC). In summary, the workflow includes the following seven steps (Fig. 1):
    Fig. 1

    TruSeq Custom Amplicon Library preparation workflow (modified by Illumina, CATALOG: FC-130-9005DOC)

    1. (a)
      Hybridization of oligo-pool (Total duration: 1 h 35 min). During this step, a custom pool containing upstream and downstream oligos specific to your targeted regions of interest is hybridized to your genomic DNA samples (see Note 1 ).

      Procedure

      1. 1.

        Make sure that concentrations and purity of your DNA samples are in agreement with those shown in the following table.

        Type of DNA

        Supported amplicon size

        Input (Up to 15 μl)

        A260/A280

        High-quality genomic DNA

        150, 175, 250, 425 bp

        50 ng (minimum)

        250 ng (recommended)

        1.8–2.0

         
      2. 2.

        Use the provided controls of TruSeq Custom Amplicon Library Preparation Kit (ACD1/ACP1) in each batch of samples and add 5 μl of control DNA and 5 μl of TE or water to 1 well in the plate.

         
      3. 3.

        To each sample well to be used in the assay, add up to 15 μl of Genomic DNA (250 ng total).

         
      4. 4.

        Add 5 μl of control oligo pool ACP1 to the well containing control DNA ACD1.

         
      5. 5.

        Using a multichannel pipette, add 5 μl of CAT to the wells containing genomic DNA (see Note 2 ).

         
      6. 6.

        Using a multichannel pipette, add 35 μl of OHS2 (Oligo Hybridization for Sequencing 2) to each sample in the plate. When dispensing, gently pipette up and down 3–5 times to mix.

         
      7. 7.

        Seal the plate with adhesive aluminum foil and secure the seal with a rubber roller or sealing wedge.

         
      8. 8.

        Centrifuge to 1,000 × g at 20 °C for 1 min.

         
      9. 9.

        Place the plate in the preheated block at 95 °C and incubate for 1 min.

         
      10. 10.

        While the plate remains on the preheated block, set the temperature to 40 °C and continue incubating for 80 min.

         
       
    2. (b)
      Removal of unbound oligos (Total duration: 20 min). This process removes unbound oligos from genomic DNA using a filter capable of size selection. Two wash steps using SW1 (Illumina) reagent ensure complete removal of unbound oligos. A third wash step using UB1 (Illumina) buffer removes residual SW1 and prepares samples for the extension ligation step.

      Procedure

      1. 1.

        Assemble the filter plate assembly unit in the following order (from top to botton): lid, filter plate, adapter collar and MIDI plate. Using a multichannel pipette, add 45 μl of SW1 (Stringent Wash 1) to each well. Cover the plate with the filter plate lid and centrifuge at 2,400 × g at 20 °C for 10 min.

         
      2. 2.

        After the 80 min incubation, confirm that the heat block has cooled to 40 °C. Remove the plate from the heat block and centrifuge to 1,000 × g at 20 °C for 1 min to collect condensation.

         
      3. 3.

        Using a multichannel pipette set to 65 μl, transfer the entire volume of each sample onto the center of the corresponding prewashed wells of the filter plate. Change tips after each column to avoid cross contamination.

         
      4.  4.

        Cover the plate with the filter plate lid and centrifuge at 2,400 × g at 20 °C for 2 min.

         
      5.  5.

        Wash the plate as using a multichannel pipette, adding 45 μl of SW1 to each sample well.

         
      6.  6.

        Cover the plate with the filter plate lid and centrifuge at 2,400 × g for 2 min (see Note 3 ).

         
      7.  7.

        Repeat the wash (steps 5 and 6).

         
      8.  8.

        Discard all the flow-through (containing formamide waste and unbound oligos) collected up to this point in an appropriate hazardous waste container, then reassemble the filter plate.

         
      9.  9.

        Using a multichannel pipette add 45 μl of UB1 (Universal Buffer 1) to each sample well.

         
      10. 10.

        Cover the plate with the filter plate lid and centrifuge at 2,400 × g for 2 min.

         
       
    3. (c)
      Extension-Ligation of bound oligos (Total duration: 50 min). This process connects the hybridized upstream and downstream oligos. A DNA polymerase extends from the upstream oligo through the targeted region, followed by ligation to the 5′ end of the downstream oligo using a DNA ligase. The extension-ligation results in the formation of products containing the targeted regions of interest flanked by sequences required for amplification.

      Procedure

      1. 1.

        Using a multichannel pipette, add 45 μl of ELM4 (Extension-Ligation Mix 4) to each sample well of the plate.

         
      2. 2.

        The extension-ligation reaction takes place on the filter plate membrane.

         
      3. 3.

        Seal the plate with adhesive aluminum foil, and then cover with the lid to secure the foil during incubation. Incubate the plate in the preheated 37 °C incubator for 45 min.

         
      4. 4.

        While the plate is incubating, prepare the Indexed Amplification Plate (IAP) as described in the following section (step 3 of PCR amplification section).

         
       
    4. (d)
      PCR amplification (Total duration: 85–105 min). In this step, the extension-ligation products are amplified using primers that add sample multiplexing index sequences as well as common adapters required for cluster generation.

      Procedure

      1. 1.

        Prepare fresh 50 mM NaOH.

         
      2. 2.

        Remove PMM2 and the index primers (i5 and i7) from −15 to −25 °C storage and thaw on a bench at room temperature.

         
      3. 3.

        Using a multichannel pipette, add 4 μl of i5 and i7primers to each column of the IAP plate (see Note 4 ).

         
      4. 4.

        For 96 samples, add 56 μl of TDP1 (TruSeq DNA Polymerase 1) to 2.8 ml of PMM2 (PCR Master Mix 2). Invert the PMM2/TDP1 PCR master mix 20 times to mix well. You will add this mix to the IAP plate in the following step.

         
      5. 5.

        When the 45 min extension-ligation reaction is complete, remove the filter plate from the incubator. Remove the aluminum foil seal and replace with the filter plate lid (see Note 5 ).

         
      6. 6.

        Centrifuge the filter plate at 2,400 × g for 2 min.

         
      7. 7.

        Using a multichannel pipette, add 25 μl of 50 mM NaOH to each sample well (see Note 6 ) and incubate the plate at room temperature for 5 min.

         
      8. 8.

        While the plate is incubating, use a multichannel pipette to transfer 22 μl of the PMM2/TDP1 PCR master mix to each well of the IAP plate containing index primers.

         
      9. 9.

        Transfer 20 μl of samples eluted from the filter plate to the corresponding column of the IAP plate. Gently pipette up and down 5–6 times to combine the DNA with the PCR master mix. Tips must be changed after each column to avoid index and sample cross contamination.

         
      10. 10.

        Cover the IAP plate with microseal film and centrifuge to 1,000 × g at 20 °C for 1 min.

         
      11. 11.
        Perform PCR on a thermal cycler using the following program and the recommended number (X) of PCR cycles:
        • 95 °C for 3 min
          • X cycles of:
            • 95 °C for 30 s

            • 66 °C for 30 s

            • 72 °C for 60 s

          • 72 °C for 5 min

          • Hold at 10 °C

         
      12. 12.

        The following table contains amplicon size, number of amplicons in your CAT (Custom Amplicon Tube), type of DNA input, and DNA input quantity to help you calculate the number of PCR cycles required.

        Amplicon size DNA input (50–99 ng)

        150/175 bp

        250 bp

        425 bp

        Number of PCR cycles (X)

        <96 amplicons

        32

        33

        33

        97–384 amplicons

        28

        28

        29

        385–768 amplicons

        26

        27

        28

        769–1,536 amplicons

        25

        26

        27

        Amplicon size DNA input (100–250 ng)

        150/175 bp

        250 bp

        425 bp

        Number of PCR cycles (X)

        <96 amplicons

        29

        30

        30

        97–384 amplicons

        25

        25

        26

        385–768 amplicons

        23

        24

        25

        769–1,536 amplicons

        22

        23

        24

         
       
    5. (e)
      PCR cleanup (Total duration: 50 min). This process uses AMPure XP beads (Beckman Coulter) to purify the PCR products from the other reaction components.

      Procedure

      1. 1.

        Prepare fresh 80 % ethanol from absolute ethanol.

         
      2. 2.

        After PCR amplification step, to confirm that the library successfully amplified, run an aliquot of the control and selected test samples on a 4 % agarose (5 μl) or on a Bioanalyzer (1 μl).

         
      3. 3.

        Prior to use, allow the beads to come to room temperature and vortex the beads until they are well dispersed.

         
      4. 4.

        Using a multichannel pipette, add the appropriate volume of AMPure XP beads (Beckman Coulter), corresponding to your amplicon size (60 μl for 150–175 bp; 45 μl for 250 bp and 35 μl for 425 bp).

         
      5. 5.

        Using a multichannel pipette set to 60 μl, transfer the entire PCR product from the IAP plate to a new plate.

         
      6. 6.

        Seal and shake the plate at 1,800 rpm for 2 min. Incubate at room temperature without shaking for 10 min.

         
      7. 7.

        Place the plate on a magnetic stand (Beckman Coulter) for 2 min or until the supernatant has cleared and with a multichannel pipette set to 100 μl, carefully remove and discard the supernatant. Change tips between samples.

         
      8. 8.

        Wash the beads with freshly prepared 80 % ethanol for two times.

         
      9. 9.

        Remove the plate from the magnetic stand and allow the beads to air-dry for 10 min.

         
      10. 10.

        Using a multichannel pipette, add 30 μl of EBT (Elution Buffer with Tris) to each well.

         
      11. 11.

        Seal and shake the plate at 1,800 rpm for 2 min.

         
      12. 12.

        Incubate at room temperature without shaking for 2 min.

         
      13. 13.

        Place the plate on the magnetic stand for 2 min or until the supernatant has cleared.

         
      14. 14.

        Using a P20 multichannel pipette and fine tips, carefully transfer 20 μl of the supernatant from the plate to a new MIDI plate and then centrifuge to 1,000 × g for 1 min.

         
       
    6. (f)
      Library normalization (Total duration: 1 h 20 min). This step normalizes the quantity of each library to ensure more equal library representation in your pooled sample.

      Procedure

      1. 1.

        Prepare fresh 0.1 N NaOH.

         
      2. 2.

        For 96 samples, add 4.4 ml of LNA1 (Library Normalization Additives 1) to a fresh 15 ml conical tube.

         
      3. 3.

        Use a P1000 pipette set to 1,000 μl to resuspend LNB1 (Library Normalization Beads 1) thoroughly by pipetting up and down 15–20 times, until the bead pellet at the bottom is resuspended, and transfer 800 μl of LNB1 to the 15 ml conical tube containing LNA1.

         
      4. 4.

        Using a multichannel pipette, add 45 μl of the combined LNA1/LNB1 to each well of the MIDI plate containing libraries.

         
      5. 5.

        Seal and shake the MIDI plate on a microplate shaker at 1,800 rpm for 30 min.

         
      6. 6.

        Place the MIDI plate on a magnetic stand for 2 min or until the supernatant has cleared and using a multichannel pipette set to 80 μl remove the supernatant.

         
      7. 7.

        Remove the plate from the magnetic stand and wash the beads with 45 μl of LNW1 (Library Normalization Wash 1) for two times.

         
      8. 8.

        Remove the plate from the magnetic stand and add 30 μl of 0.1 N NaOH

         
      9. 9.

        Seal and shake the plate on a microplate shaker at 1,800 rpm for 5 min.

         
      10. 10.

        During the 5 min of elution, prepare a new 96-well PCR plate and add 30 μl LNS2 (Library Normalization Storage buffer 2) to each well.

         
      11. 11.

        Using a multichannel pipette set to 30 μl, transfer the supernatant from the first plate to the second plate. Change tips between samples to avoid cross contamination.

         
      12. 12.

        Seal the plate and then centrifuge to 1,000 × g for 1 min (see Note 7 ).

         
       
    7. (g)
      Library pooling and MiSeq sample loading (Total duration: 10 min). In preparation for cluster generation and sequencing, equal volumes of normalized library are combined, diluted in hybridization buffer, and heat denatured prior to sequencing on the MiSeq.

      Procedure

      1. 1.

        Determine the samples to be pooled for sequencing, based on the number of targeted regions and desired coverage, using the following table (Illumina; CATALOG: FC-130-9005DOC) [11].

        Amplicons per CATa

        Desired mean coverageb

        Suggested maximum Samples per MiSeq run

         

        MiSeq v2b

        MiSeq v3b

        16

        150×

        96

        96

        500×

        96

        96

        48

        150×

        96

        96

        500×

        96

        96

        96

        150×

        96

        96

        500×

        96

        96

        384

        150×

        96

        96

        500×

        48

        80

        768

        150×

        72

        96

        500×

        24

        40

        1,536

        150×

        36

        60

        500×

        12

        20

        aCustom Amplicon Tube; bMiSeq Reagent Kit version

         
      2. 2.

        Using a P20 multichannel pipette, transfer 5 μl of each library to be sequenced from the plate, column by column, to a PCR eight-tube strip.

         
      3. 3.

        Combine and transfer the contents of the PCR eight-tube strip into a fresh Eppendorf tube.

         
      4. 4.

        Create your Diluted Amplicon Library by combining the volumes of HT1 (Hybridization buffer) and Pooled Amplicon Library based on your MiSeq Reagent Kit version.

         
      5. 5.

        Mix Diluted Amplicon Library by vortexing the tube and using a heat block, incubate the tube at 96 °C for 2 min.

         
      6. 6.

        After the incubation, invert the tube 1–2 times to mix and immediately place in the ice water bath for 5 min.

         
      7. 7.

        Load Diluted Amplicon Library into a thawed MiSeq reagent cartridge in the Load Samples reservoir.

         
       
     

3.4 Sequence Libraries on MiSeq and Analyze Data

  1. 1.

    In parallel to library preparation, a sample sheet, to identify each sample and its corresponding index, should be prepared. To prepare your sample sheet, use the Illumina Experiment Manager, a wizard-based application that allows the recording of your sample ID, workflow, indices, and other parameters applicable to your 96-well plate. The Illumina Experiment Manager can be run on any Windows platform. You can download the Illumina Experiment Manager from the Illumina website.

     
  2. 2.

    TruSeq Custom Amplicon Library must be sequenced on a MiSeq sequencing system. For more details on using the MiSeq instrument or setting up your run, see the MiSeq System User Guide (Illumina, part # 15027617).

     
  3. 3.

    Analyze data with MiSeq Reporter software. MiSeq Reporter processes the base calls generated by the MiSeq sequencing system. It is an on-instrument software and produces information such as alignment and structural variants. For TruSeq Custom Amplicon libraries, it produces aligned reads in the BAM format and outputs variant calls in .vcf files. For more information, see the MiSeq System User Guide or MiSeq Reporter’s online help (www.illumina.com/help/miseq_reporter/default.htm).

     
  4. 4.

    Analyze data with Illumina Amplicon Viewer. The Illumina Amplicon Viewer has been designed and developed for off-instrument visualization and analysis of TruSeq Custom Amplicon data. Amplicon Viewer allows you to view data (including coverage, Q-score, variant calls, etc.) from multiple MiSeq amplicon runs simultaneously and interactively. DesignStudio might need to cover a large contiguous region with multiple amplicons, and the Illumina Amplicon Viewer opens the reconstituted contiguous region with combined coverage and variants appropriately. You can also export custom reports based on selected samples/targets/variants.

     
  5. 5.

    Annotation of variants should be performed using 1000Genomes and NCBI and ESP6400 databases by GATK2 tool or using Variant Studio software (Illumina).

     
  6. 6.

    Previously unreported DNA variants should be evaluated: (1) by looking at their frequency in controls; (2) by looking at segregation; (3) by comparative sequence analysis among different species; (4) by applying different prediction softwares (PolyPhen, Fruitfly, Genscan, ESE finder).

     

4 Notes

  1. 1.

    To ensure consistency across samples, use a multichannel pipette where possible. Calibrate pipettes periodically.

     
  2. 2.

    Always use fresh pipette tips between samples and between dispensing index primers.

     
  3. 3.

    If the SW1 does not drain completely after 2 min, the plate can be centrifuged again for up to 10 min. Significantly incomplete drainage of SW1 compromises target enrichment specificity.

     
  4. 4.

    Tips must be changed after each row to avoid index cross contamination.

     
  5. 5.

    Removing the aluminum foil seal before centrifugation is recommended to ensure the reaction supernatant drains into the waste plate effectively.

     
  6. 6.

    Ensuring that pipette tips come in contact with the membrane, pipette the NaOH up and down 5–6 times. Tips must be changed after each column.

     
  7. 7.

    The final library pool consists of single-stranded DNA, which does not resolve well on an agarose gel or Bioanalyzer chip. qPCR can be used for quality control if desired.

     

Notes

Acknowledgments

This work was supported in part by grant “Premio di ricerca Gianluca Montel, 2011/2012” from University of Foggia, awarded to Maddalena Gigante and Progetto Strategico Regione Puglia grant (E.R., 2008), Ministero dell’Istruzione, dell’Università e della Ricerca (MiUR) FIRB, CAROMICS grant (E.R., 2011). The authors thank Illumina Fiel Application Scientist for technical support.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Maddalena Gigante
    • 1
    • 2
    Email author
  • Sterpeta Diella
    • 1
    • 2
  • Elena Ranieri
    • 1
    • 2
  1. 1.Department of Medical and Surgical SciencesUniversity of FoggiaFoggiaItaly
  2. 2.Ospedali RiunitiFoggiaItaly

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