Assay primers design
Assay primers were designed for all regions of interest using Oligo, NCBI/SNPdb, NCBI/BLAST, Ensembl, and other computer aided design tools as well as SeqScape and Sequencher for an optimum annealing temperature of 58-60°.
Assay storage and distribution to 384-well plates
Primers were received from the vendor in a 2.0 mL vial in a lyophilized state with the antisense (Coding, Forward or Upper) primer and the sense (Non-coding, Reverse or Lower) primer in separate tubes. Primers were suspended to 60 μM in warm 25% glycerol and 75% nuclease free de-ionized water. From these stocks, working assays were prepared in 1.4 mL ScreenMate™ tubes (PN 4247) with SepraSeal™ caps PN 4464 (Matrix Technology Corp. Hudson, N.H.) using 960 μL of 25% glycerol and 20 μL of forward primer 60 μM stock and 20 μL of reverse primer 60 μM stock. This resulted in a solution of 1.2 μM each or 2.4 μM total primer concentration in 25% glycerol. The Hamilton MicroLab StarPlus distributed 6 μL of this assay solution into the wells of a 384-well plate and the plate was dried (70°C for 40 min.) to 1.5 μL of pure glycerol at 4.8 μM each or 9.6 μM total primer concentration. Similarly, the M13F (5'-TGTAAAACGACGGCCAGT-3') and M13R (5'-CAGGAAACAGCTATGACC-3') primers were ordered and received in 2.0 mL vials in a lyophilized state in separate tubes and suspended to 60 μM in warm 25% glycerol and 75% nuclease free de-ionized water. From these stocks working concentrations at 1.8 μM in 25% glycerol were prepared. All stocks in 25% glycerol can be stored for short term at 4°C if tightly capped (and perhaps wrapped in parafilm) and at -20°C if stored for longer than 4 weeks. The Hamilton MicroLab StarPlus distributed 2 μL of forward or reverse sequencing primer into all wells of a 384-well plate (one plate for M13F and one plate for M13R), and the plates were dried (70°C for 20 min.) to 0.5 μL of pure glycerol at 7.2 μM concentration. All plates that contain primers diluted in pure glycerol may be sealed and stored at room temperature for future use or used immediately. 1.5 μL of glycerol will not adversely affect a 12 μL total volume PCR, nor will 0.5 μL of glycerol adversely affect a 10 μL total volume cycle sequencing reaction.
PCR reaction assembly
PCR reactions were assembled in a 384-well plate (AB1111, Thermo Fisher Scientific Inc., Waltham, MA) automatically using a Hamilton MicroLab StarPlus liquid handling/pipetting system.
DNA extraction and distribution to 384-well plates
DNA was extracted and purified from saliva, blood or tissue according to standard protocol from vendors (e.g. Oragene™, DNA Genotek Inc., Ottawa, Ontario, Canada; Gentra Puregene™, QIAGEN Inc. Valencia, CA). As currently configured, 16.0 μg is required to complete all five tiers of testing. Genomic DNA was diluted first to 100 ng/μL in nuclease free ddH2O and then to 10 ng/μL using 1× PCR buffer.
The Hamilton MicroLab StarPlus distributed 3 μL of sample DNA to the appropriate wells of the 384-well PCR plate that already contains assays in 1.5 μL glycerol (Additional files 44 and 45).
Master Mix preparation and distribution
A master mix was prepared consisting of 5.17 μL ddH2O (nuclease free), 0.93 μL 10× buffer (0.5 M Tris HCl, 0.1 M KCl, 0.05 M (NH4)2SO4 and 0.0208 M MgCl2 PH 8.1), 1.2 μL Bioline dNTPs 2.5 mM each dNTP (part number BIO-39025 BioLine USA, Taunton, MA) and 0.2 μL Roche FastStart™ polymerase 1 Unit (Part number 12 032 945 001 Roche Diagnostic GmbH, Mannheim, Germany) totalling 7.5 μL per 12 μL PCR reaction. PCR was performed in an ABI 9700 thermal cycler under the following conditions: initial denaturation and enzyme activation at 95.0°C for 1 cycle of 6 min, followed by 35 cycles of denaturation at 95.5°C for 55 s, annealing at 58.0°C for 30 s, and extension at 72.0°C for 55 s followed by a final single cycle extension at 72.0°C for 7 min.
Post PCR processing
After PCR, the Hamilton MicroLab StarPlus added 5 μL of 1× PCR buffer to each well to ensure there was enough volume for the QIAxcel system, and the 384-well plate was distributed to four 96-well plates since the QIAxcel system currently only handles 96-well plates. After visually searching the gel image or spreadsheet output of the QIAxcel system to detect any deletions greater than 14 bp or duplications greater than 14 bp (but less than PCR product size), product concentrations were determined. The Hamilton MicroLab StarPlus condensed the four plates back to a 384-well plate for magnetic bead purification and sequence reaction assembly. PCR purification was per AMPure™ protocol (Agencourt Biosciences Corp. Beverly, MA) slightly modified for the Hamilton robot. Using concentration values from the QIAxcel system in tab delimited format, PCR reactions were selectively diluted by the Hamilton MicroLab StarPlus to achieve normalization of concentration to a range from 2 to 7 ng/μL. Forward and reverse sequence reactions were assembled separately in two 384-well plates. A 1/16th Big Dye™ (Applied Biosystems Inc. part of Life Technologies, Carlsbad, CA) master mix was prepared using: 0.34 μL Big Dye, and 2.41 μL 5× Big Dye Buffer. 2.75 μL master mix was pipetted into the wells of the 384 well sequencing plates which already contained the sequencing primer in 0.5 μL pure glycerol. Next 6.75 μL of the purified and normalized PCR product were pipetted into the appropriate wells. Cycle sequencing was performed on the 10.0 μL sequencing reaction in an ABI 9700 thermal cycler under an initial denaturation at 96.0°C for 4 min followed by 25 cycles of denaturation at 96.0°C for 10 s, annealing at 50.0°C for 5 s, and extension at 60.0°C for 4 min. Sequence reaction purification was accomplished per CleanSeq™ protocol (Agencourt Biosciences Corp. Beverly, MA) also slightly modified for the Hamilton automation. After CleanSeq™, the purified sequencing product plates were heat-sealed and installed in 3730 sequence assembly plates (Applied Biosystems) with Direct Inject Magnets (Agencourt) and loaded into the 3730 sequencer. An ABI 3730 sequencer was used to perform the sequencing operation and Mutation Surveyor and/or SeqScape and/or Sequencher applications were used to analyze the resulting sequences and to search for variations from a consensus sequence.
Cost Estimates
Table 8 presents an estimate of reagent and plastic costs based on 12 uL PCR and 10 uL sequencing reaction volumes.
One approach to pricing the service would be to average the price per patient over a typical mix of 100 muscular dystrophy patients across all five tiers of testing. Consumable material costs for MLPA (tier 1 = 2 MLPA kits) including plastics and 20% waste are approximately $48.00 ($24.00/kit) and for tier 5 including a much larger number of reference samples is approximately $40.00 per kit. Labor cost is estimated at 2 hours per patient of dedicated technician time for tier 1, 8 hours per patient of one technician for each tier 2-4 - primarily for sample processing in steady state continuous mode - plus 8 hours per patient of a second dedicated technician primarily for sequence analysis, and finally 1 hour per patient per MLPA kit for tier 5. Using those cost estimates, the percentage of DMD incidence (80%, use 60% to be conservative) versus total dystrophies and the percentage of large deletion/duplication (60%) in DMD/BMD cases, and $20.00/hour labor rate, average the direct cost per patient over 100 patients as:
Consumable material costs:
Extract DNA from blood or saliva: 100 × $15.00/patient = $1,500.00
Tier 1 (2-MLPA-rxns.): 100 × $48.00/patient = $4,800.00
Tier 2: 64 non DMD/BMD large del/dup × $532.00 Plate 1 = $34,048.00
Tier 3: 28 remaining undetected × $395.00 Plate 2 = $11,060.00
Tier 4: 18 remaining undetected × $395.00 Plate 3 = $7,110.00
Tier 5: 8 remaining undetected × $40.00 × 4 MLPA's = $1,280.00
Total consumables = $59,798.00/100 = $600.00/patient
Add direct labor:
Extract DNA: 100 patients × 2 hrs./patient × $20.00/hr = $4000.00
Tier 1: 100 patients × 2 hrs./patient × $20.00/hr. = $4,000.00
Tier 2: 64 patients × 16 hrs./patient. × $20.00/hr. = $20,480.00
Tier 3: 28 patients × 16 hrs./patient × $20.00/hr. = $8,960.00
Tier 4: 18 patients × 16 hrs./patient × $20.00/hr. = 5,760.00
Tier 5: 8 patients × 5 hrs./patient × $20.00/hr. = $800.00
Total labor = $44,000.00/100 = $440.00/patient
Total consumables plus labor = $1040.00/patient
Add to this whatever your cost structure requires for indirect/overhead including instrument maintenance costs+ profit + taxes = final price per patient direct cost. Even if that final service pricing cannot be structured this way for various reasons, these numbers still provide a good estimate of the average cost per patient to the department.
Clinicians can significantly decrease the actual cost by steering the molecular testing to a candidate gene based on phenotype, clinical evaluations and any immunolabeling data that has been prepared.
Quality Goals
Two quality goals were set for assays as follows: 1) No known multiple nucleotide polymorphisms (MNPs) or SNPs within primer sequences except rare SNPs allowed in 5' half of primer. 2) A 12 μL PCR increased to 17 μL with 1×-PCR buffer will have a single sharp band of the planned size and a minimum concentration 0.5 ng/μL (preferably > 2.0 ng/μL) when run on QIAxcel system DNA analyzer (Figure 2).
Three goals were set for gene coverage as follows: 1) All regions of interest will be covered. Regions of interest are pre-defined for each gene and do vary but usually consist of all coding and non-coding exons, splice sites, promoters, 1000 bp of 5' UTR and 3000 bp of 3' UTR. 2) At least 95% of the total sequence for all regions of interest of a given gene is covered by both the sense and anti-sense sequencing reaction. Difficult regions to sequence such as polynucleotide repeats and G/C rich areas will have multiple assays attempting to sequence through the region and will have at least single-stranded coverage. 3) For any given patient, the quality score for every pure (i.e. homozygous) base (as calculated by SeqScape and based on the quality scores of each strand running through that base) for all bases except those in a very few clearly difficult to sequence regions must be greater than 25 on the 0-50 scale used by SeqScape.
Patient materials and mutation reporting
This study was approved by the Children's Hospital Boston Institutional Review Board. Written informed consent was obtained for each participant. Mutations found using this process should be reported to the Leiden muscular dystrophy pages with technique described as PCR, SEQ.