SMaRT M-Seq: an optimized step-by-step protocol for M protein sequencing in monoclonal gammopathies

A. MNC separation and lysis

The starting material of the protocol is represented by primary mononuclear cells (MNCs), which are isolated by a standard density gradient centrifugation of bone marrow (BM) aspirate (refer to step 1 of Fig. 1). The procedural steps in performing a BM aspiration have been described in detail previously [27], and a video demonstrating the procedure for BM aspiration from the posterior iliac crest is available [28]. The MNCs obtained through this process are subsequently lysed in TRIzol and stored at −80°C. This aliquot will then be used for RNA extraction (Section B). Refer to the note at the end of this section for alternatives to TRIzol.

Caution: Work under a biological safety cabinet due to the use of hazardous samples (human BM aspirate) and toxic reagents (TRIzol).

1. Starting from BM aspiration, transfer the sample into a 15-mL or 50-mL conical tube and dilute it with two volumes of DPBS;

2. Considering the obtained volume, carefully stratify the diluted sample in a 1:1 proportion in a 15-mL or 50-mL conical tube containing Lympholyte–H (Fig. 2A);

3. Centrifuge for 30 minutes at 450 × g at room temperature in a swinging bucket rotor with brake off;

4. If present, carefully remove and discard any visible tissue particles floating in the top layer of the sample, to avoid aspirating them during the collection of the MNC layer.

5. Slowly introduce the serological pipette inside the tube to reach the MNC layer (Fig. 2A) and carefully aspirate the MNC layer, paying attention not to aspirate any Lympholyte-H, and transfer it into a new 15-mL or 50-mL conical tube;

6. To remove any residual Lympholyte-H, wash the MNC layer by filling the 15-mL or 50-mL conical tube containing the MNC layer with FACS buffer;

7. Centrifuge for 10 minutes at 450 × g at 4°C, with brake on;

8. Discard the supernatant and resuspend the MNCs pellet in 10 mL of FACS buffer and filter the cell suspension by using a sterile 70-μm cell strainer;

9. For MNCs counting, pipette 10 µL of the cell suspension and 90 µL of Trypan Blue (1:10 dilution) in a 1.5-mL conical microtube and mix thoroughly;

10. Pipette 10 µL of this mixture into a cell counter chamber and count the number of MNCs within two non-consecutive squares of the grid using a light microscope (e.g. counting the upper left and bottom right squares). If cells are highly concentrated, dilute the cell suspension (obtained in step 8) in 20 mL of FACS buffer;

11. Divide the number of cells counted by the number of counted squares (2); multiply the obtained number by the dilution factor (i.e. 10) and multiply by 10⁴ (hemacytometer factor) to calculate the number of cells per mL of cell suspension. The total number of MNCs is finally obtained multiplying the number of cells per mL (the concentration) by the number of mL of the cell suspension (total volume, i.e. 10 mL);

12. Take an aliquot corresponding to 1x10⁷ cells;

13. Centrifuge for 10 minutes at 450 × g at 4°C, and lyse the cell pellet in 1 mL of TRIzol;

14. Transfer the lysate in a 1.5-mL tube and store the sample at −80°C.

Pause point: The TRIzol sample can be stored at −80°C for several months.

Critical: Acceleration/deceleration braking ramps should be set at the minimum to enable slow acceleration/deceleration and favor the formation and maintenance of the density gradient.

Note: To improve the quality of the MNC isolation procedure, it is possible to strain the BM sample using a sterile 70-μm cell strainer, to remove possible tissue particles or clots, before diluting the sample with DPBS (step 1).

Note: Lympholyte–H is selected due to its ability to provide a MNC layer devoid of granulocytes. If substituting with an alternative commercially available reagent, ensure that this specific property is retained and adapt the subsequent steps of gradient centrifugation following the manufacturer’s instructions.

Note: Due to the deactivation of the acceleration and brake functions, the actual centrifugation time of step 3 is higher than 30 minutes.

Note: As an alternative method for MNC layer collection (step 5), discard the plasma: DPBS fraction by aspiration and then collect the MNC layer using a serological pipette.

Note: If less than 1x10⁷ MNCs are retrieved, lyse all the obtained cells in a single TRIzol aliquot.

Note: As an alternative to TRIzol-based MNCs lysis, storage and subsequent RNA extraction, commercially available RNA extraction kits (e.g. RNeasy Mini kit, QIAGEN, catalog number: 741049; RNeasy Plus Micro kit, QIAGEN, catalog number: 74034) can be used. Lyse the MNC aliquot (obtained in step 13) in the lysis buffer provided by the selected kit and follow the manufacturer’s instructions for RNA extraction.

B. RNA extraction by TRIzol

The TRIzol aliquot of MNCs obtained from the BM sample is used for the RNA extraction (refer to step 2 of Fig. 1). The obtained RNA will serve as the input material for double-stranded cDNA (dscDNA) synthesis (Section C). If the MNCs were lysed using a reagent other than TRIzol (step 13), proceed with RNA extraction according to the selected kit and continue the protocol from step 27.

Caution: Work under a chemical hood due to the toxicity of TRIzol and chloroform.

• 15.

  Thaw the TRIzol aliquot of MNCs on ice;

• 16.

  Incubate for 5 minutes on ice to ensure complete dissociation of the nucleoprotein complex;

• 17.

  Add 200 µL of chloroform for each mL of TRIzol used, vortex vigorously for 15 seconds, and incubate for 2–3 minutes;

• 18.

  Centrifuge the sample for 15 minutes at 12,000 × g at 4°C;

• 19.

  Transfer the aqueous phase into a new 1.5-mL tube, taking care not to aspirate the phases below (Fig. 2B);

Note: Alternatively, after step 19 RNA extraction can be performed using RNeasy Mini kit (QIAGEN, catalog number: 741049) or RNeasy Plus Micro kit (QIAGEN, catalog number: 74034). Choose the kit according to the number of MNCs lysed in TRIzol. For this purpose, add 1 volume of 100% ethanol to the aqueous phase and briefly vortex the sample. Proceed with RNA extraction following the manufacturer’s instructions from step 3 of the RNeasy Mini Kit (version November 2021—transferring the sample in the RNeasy Mini spin column) and step 4 of the RNeasy Plus Micro Kit (version March 2016—transferring the sample in the RNeasy MinElute spin column).

Pause point: The sample can be stored at −20°C overnight.

• 20.

  Add 500 µL of isopropanol to the aqueous phase, vortex, and incubate for 10 minutes on ice;

• 21.

  Centrifuge for 10 minutes at 12,000 × g at 4°C;

• 22.

  Remove and discard the supernatant, keeping the tube on ice. The total RNA precipitate is visible as an opaque white pellet at the bottom of the tube;

• 23.

  Overlay the pellet with 1 mL of cold 75% ethanol;

• 24.

  Centrifuge for 5 minutes at 7,500 × g at 4°C;

• 25.

  Carefully remove and discard the supernatant by aspirating it with a micropipette;

Critical: The pellet obtained after the centrifugation performed in step 22 is not always clearly visible. To ensure the pellet is not lost, centrifuge the sample placing the tube in the centrifuge in a known orientation. Then, after the centrifugation, aspirate the supernatant using a tip directed opposite to where the pellet is expected to be. Ethanol residue can cause RNA degradation. Allow the ethanol to evaporate for 10 minutes to avoid this problem.

• 26.

  Gently resuspend the pellet in 20–50 µL of DNase- and RNase-free water;

• 27.

  Assess the RNA quality and yield by Nanodrop spectrophotometer;

Note: It is recommended to assess the integrity of the extracted RNA by agarose gel electrophoresis or capillary electrophoresis before proceeding with dscDNA synthesis (Fig. 3).

Pause point: RNA samples can be stored at −80°C for several months.

C. Synthesis, purification, and precipitation of dscDNA

In this step, RNA is retrotranscribed into dscDNA. The retrotranscription process involves priming with an anchored oligonucleotide, followed by the synthesis of the first strand. Subsequently, it proceeds with second strand synthesis through various enzymatic reactions to generate dscDNA. The dscDNA will then be subjected to circularization to obtain ligcDNA (Section D) (refer to step 3 of Fig. 1).

Caution: During the entire duration of this procedure, work on ice with the exception of steps 33 and 36. Before each incubation, vortex/flick the sample and briefly centrifuge it to spin it down. After each incubation, briefly centrifuge the sample to spin it down and place the tube on ice.

• 28.

  Transfer a volume corresponding to 500–1000 ng of RNA into a new tube and adjust the final volume to 10 µL by adding DNase- and RNase-free water if necessary;

Note: In poorly concentrated samples, where 10 µL of RNA contains less than the minimum RNA concentration required for dscDNA synthesis (500 ng), a carrier RNA can be used. For this purpose, use 9 µL of RNA sample and 1 µL of carrier RNA (possibly concentrated 500 ng/µL). Carrier RNA should not include immunoglobulin gene transcripts to prevent interference with the downstream analysis. RNA from HeLa cells can be conveniently used for these purposes. For alternative sources of carrier RNA, it is recommended to first verify that said RNA, upon retrotranscription, dscDNA synthesis and circularization, does not result in any amplicon formation after inverse PCR (see below).

• 29.

  Add the following reagents into the 1.5-mL tube:

• 1 µL of Anchored Oligo-dT (diluted as 0.5 µg/µL, by using DNase- and RNase-free water)

• 1 µL of RNaseOUT enzyme

• 30.

  Incubate for 10 minutes at 70°C;

• 31.

  Briefly spin down the sample to remove the droplets from the cap, and add the following reagents:

• 4 µL of 5X First strand reaction buffer

• 2 µL of 0.1 M DTT

• 1 µL of dNTPs mix (diluted and mixed as 10 mM stock, by using DNase- and RNase-free water)

• 32.

  Incubate for 2 minutes at 45°C to equilibrate the temperature;

• 33.

  Keeping the sample out of ice, add 1 µL of Superscript II Reverse Transcriptase and incubate for 1 hour at 45°C. The total volume of the reaction for the synthesis of the first strand of cDNA is now 20 µL;

• 34.

  Briefly spin down the sample to remove the droplets from the cap, and continue with the synthesis of the second cDNA strand by adding the following reagents:

• 93 µL of DNase- and RNase-free water

• 30 µL of 5X Second Strand reaction buffer

• 3 µL of dNTP mix (diluted and mixed as 10 mM stock, by using DNase- and RNase-free water)

• 1 µL of E. coli DNA ligase

• 2 µL of E. coli DNA polymerase I

• 1 µL of E. coli DNA RNase H

The total volume of the reaction for the synthesis of the second cDNA strand is now 150 µL;

• 35.

  Incubate for 2 hours at 16°C;

• 36.

  After incubation, keeping the sample out of ice, add 1 µL of T4 DNA polymerase and continue the incubation for 5 minutes at 16°C;

Caution: After step 36, work under a chemical hood from step 37 to step 42, to avoid exposure to phenol–chloroform–isoamyl alcohol mixture (25:24:1), which is toxic.

• 37.

  Add 160 µL of Phenol-chloroform-isoamyl alcohol mixture (25:24:1), vortex, and incubate for 2–3 minutes until phase separation;

• 38.

  Centrifuge the sample for 5 minutes at 12,000 × g at 4°C;

• 39.

  Carefully collect the upper phase (approximatively 150 µL) containing the dscDNA and transfer it into a second new 1.5-mL tube, taking care not to aspirate the phase below (Fig. 2C). Close this second tube, and keep it on ice, until step 42;

• 40.

  Add 160 µL of STE buffer to the initial tube containing the lower phase with phenol–chloroform–isoamyl alcohol mixture (25:24:1). Vortex and incubate for 2–3 minutes until phase separation;

• 41.

  Centrifuge the sample for 5 minutes at 12,000 × g at 4°C;

• 42.

  Carefully collect the upper phase (approximately 150 µL) and transfer it into the second tube from step 39, taking care not to aspirate the phase below. Discard the initial tube, now containing only the lower phase. The second tube, now containing a mix of the two upper phases, should have a total volume of approximatively 300 µL;

• 43.

  Proceed with dscDNA precipitation by adding the following reagents:

• 100 µL of ammonium acetate

• 750 µL of cold 100% ethanol

• 44.

  Vortex vigorously the sample and incubate the tube on ice for 10 minutes;

• 45.

  Centrifuge for 20 minutes at 12,000 × g at 4°C;

• 46.

  Gently remove the supernatant and overlay the pellet with 500 µL of cold 70% ethanol;

• 47.

  Centrifuge for 10 minutes at 12,000 × g at 4°C;

• 48.

  Carefully remove the supernatant;

Note: The pellet obtained after the centrifugation performed in step 45 is typically not visible. To ensure the pellet is not lost, centrifuge the sample placing the tube in the centrifuge in a known orientation. Then, after the centrifugation, aspirate the supernatant using a tip directed opposite to where the pellet is expected to be. Keep the same tube orientation in the centrifuge for step 47.

Critical: Ethanol residues can cause PCR inhibition. Allow the ethanol to evaporate for 10 minutes to avoid this problem.

• 49.

  Resuspend the pellet in 10 µL of DNase- and RNase-free water;

Pause point: dscDNA can be stored at -20°C for several months.

D. Circularization of dscDNA

The synthesized dscDNA is circularized in a DNA ligase-mediated reaction (refer to step 3 of Fig. 1). This reaction is crucial to perform the inverse PCR and amplification of the immunoglobulin target region.

• 50.

  Prepare the DNA ligation reaction by adding the following reagents into a new 1.5-mL tube:

• 4 µL of DNase- and RNase-free water

• 2 µL of 5X T4 DNA ligase buffer

• 3 µL of dscDNA

• 1 µL of T4 DNA ligase

• 51.

  Incubate for 16-20 hours at 14°C;

Caveat: After incubation, briefly centrifuge the sample to spin it down and place the tube on ice.

Pause point: Circularized dscDNA (ligcDNA) sample can be store at -20°C for several months.

E. Inverse PCR and amplification of immunoglobulin target region

The circularized dscDNA (ligcDNA) is used to perform a PCR protocol consisting of two steps: a first inverse PCR allows amplification of the immunoglobulin isotype of interest and the incorporation of universal adapters (from here on termed inverse PCR; refer to step 4 of Fig. 1); a second PCR (from here on termed barcoding PCR) enables the molecular barcoding of each sample using barcoded primers annealing to the universal adapters incorporated during the inverse PCR (refer to step 5 of Fig. 1). To ensure the absence of any reagent contaminations and the correctness of the prepared PCR mixtures, it is recommended to add a negative control (H₂O) and a positive control (i.e. a plasma cell line ligcDNA), respectively. If an RNA carrier was employed for samples with low RNA yield, it is recommended to also include a ligcDNA sample from the original RNA carrier alone as an additional negative control.

• 52.

  Prepare the inverse PCR reaction mixture by adding the following reagents (per sample) to reach a final volume of 25 µL:

• 12.5 µL of Q5 High-Fidelity 2X Master Mix

• 1.25 µL of Universal target-specific forward primer (diluted as 10 µM)

• 1.25 µL of Universal target-specific reverse primer (diluted as 10 µM)

• 7 µL of DNase- and RNase-free water

• 3 µL of ligcDNA obtained from step 51

Caveat: Briefly vortex and spin down the sample to favor mixing of the reagents.

• 53.

  Proceed with amplification as shown below:

Note: See Troubleshooting section for suboptimal PCR results.

Pause point: Inverse PCR amplicons can be stored at −20°C for few days.

• 54.

  Prepare the barcoding PCR reaction mixture by adding the following reagents (per sample) to reach a final volume of 25 µL:

• 12.5 µL of Q5 High-Fidelity 2X Master Mix

• 2.5 µL of F/R barcoded primer

• 7 µL of DNase- and RNase-free water

• 3 µL of template derived from inverse PCR (step 53)

Caveat: Briefly vortex and spin down the sample to favor mixing of the reagents.

• 55.

  Proceed with amplification as shown below:

Note: See troubleshooting section for suboptimal PCR results.

• 56.

  After PCR amplification, run the PCR products on 1% agarose gel(s) in 1X TAE buffer with a DNA visualization dye other than ethidium bromide. Use every other well for sample loading to facilitate subsequent band excision. Include a 1kb DNA ladder in one lane of each agarose gel, or in one lane of each row if using a multi-row agarose gel (Fig. 4);

• 57.

  Visualize DNA bands by exposing the agarose gel to a blue light on a transilluminator. Cut bands from the gel and collect them into separate 1.5-mL tubes.

• 58.

  Purify amplicons using the MinElute Gel Extraction kit (QIAGEN) according to the manufacturer’s instructions. In the final steps of the extraction protocol, elute the amplicon from the MinElute Spin Columns by using 10 µL of DNase- and RNase-free water

• 59.

  Assess the concentration of each eluted amplicon by testing 1 µL of amplicon eluate on a Qubit fluorometer according to the manufacturer’s instructions;

• 60.

  Pool equimolar amounts of barcoded amplicons to achieve a final concentration of 1 µg;

Note: The total amount of DNA to be used for the construction of the SMRTbell library (Section F) is in accordance with PacBio guidelines.

Critical: Ethidium bromide and/or ultraviolet light can induce DNA damage and should be avoided, as DNA damage impinges subsequent SMRT. For additional sample requirements for SMRT, please refer to the “PacBio guidelines for successful SMRTbell^TM libraries” document (https://dna.uga.edu/wp-content/uploads/sites/51/2018/02/Pacbio-Guidelines-SMRTbell-Libraries-v1.0.pdf) [29].

F. SMRT bell library construction and sequencing

The pool of barcoded PCR amplicons generated in the previous steps (refer to step 6 of Fig. 1) is used to create the SMRTbell library following PacBio guidelines. In this step, SMRTbell adapters are included in the amplicons through a ligation reaction, to obtain circularized DNA amplicons (https://www.pacb.com/wp-content/uploads/SMRTbell-Library-Preparation-for-High-Fidelity-Long-Read-Sequencing-Customer-Training.pdf, https://www.pacb.com/wp-content/uploads/Procedure-Checklist-Preparing-HiFi-SMRTbell-Libraries-using-SMRTbell-Express-Template-Prep-Kit-2.0.pdf). The library is then subjected to SMRT (refer to steps 7 and 8 of Fig. 1) with circular consensus sequencing (CCS) generation.

The CCS resulting from sequencing is processed through bioinformatics analysis and demultiplexed according to PacBio guidelines, to obtain a FASTA file per sample (refer to step 9 of Fig. 1).

All the procedures described in Section F are carried out by a commercial provider.

G. Immunoglobulin analysis by immunogenetic analysis

• 61.

  FASTA files obtained from CCS demultiplexing are analyzed using the Vidjil immunogenetics software to identify the patient-specific clonal sequence in each FASTA file. When analyzing BM samples, the expressed repertoire of the immunoglobulin gene of interest is typically highly skewed toward the clonal sequence and patient-specific clonal sequences typically correspond to the first, most abundant, discrete sequence with the highest number of reads in the Vidjil analysis (refer to step 10 of Fig. 1). Vidjil tool can be accessed through the online public server (https://app.vidjil.org/), which is open-source, with accounts provided free of charge for research use only. Alternatively, Vidjil can be accessed through the VidjilNet Healthcare server (https://health.vidjil.org/) or can be In-lab/in-hospital hosted, and in this version, Vidjil is compliant for clinical use. Local restrictions may apply to the use of web-based or external servers. A tutorial for Vidjil is available online (https://www.vidjil.org/doc/tutorial/mastering-vidjil.html). For each FASTA file, create a “new patients” entry and upload the corresponding FASTA file to the Vidjil tool (https://app.vidjil.org/);

• 62.

  Start the analysis by opening the “process config” window;

• 63.

  In the “V(D)J recombinations” section, select “multi+inc+xxx analysis” (default: multi-locus, with same incomplete/unusual/unexpected recombinations) and launch the analysis by clicking on “Run all”;

• 64.

  When the analysis is completed, the “multi+inc+xxx” will appear in the results column, and by clicking on this, the result for each FASTA file can be visualized;

• 65.

  Selecting the sequenced isotype, a list of all the “clones” present in the sample and their relative abundance in terms of the number of reads is provided;

• 66.

  Sort the data obtained according to the relative frequency (“Sort by size” field) to identify the dominant clone (Fig. 5), which should display the highest percentage of equal reads than all other clones possibly present in the biological sample;

• 67.

  For each clone, the sequence information can be retrieved by clicking on the symbol “ï” next to the % of the molecular clonal size [i.e. the sequence length, theclonal size (expressed both in terms of absolute number of clonal reads out of the total reads and in terms of percentage), the nucleotide sequence, the sequence productivity, the V, J, and possibly D gene and allele assignment];

Note: the IMGT/V-QUEST portal (https://www.imgt.org/IMGT_vquest/input) can be used to further verify the productivity and the V, D, and J germline gene and allele assignment of the dominant clonal sequence (selecting the species and the sequenced isotype of interest).

Note: Alternatively to Vidjil, other immunogenetics tools such as IMGT/HighV-QUEST (https://www.imgt.org/HighV-QUEST/home.action), IgBlast (https://www.ncbi.nlm.nih.gov/igblast/), and MiXCR can be used.