Cost of gene panels sequencing

Why do you do this experiment?

Gene panels are curated sets of genes with known significance for a specific disease or collection of clinical symptoms.

Input 100ng genomic DNA (~100k cells)

Output Fastq file (100k SE reads) -> High depth sequence of the genes in the panel

Strategic Value

• Elucidate the cause of a genetic disease.
• Detect subclonal mutations to adapt treatment before the resistant clones cause a relapse (from biopsy or circulating tumor DNA).

Cost & Scale

• Variable per run: \$58/sample with range \$61 (sequenced on large sequencer with other samples) - \$113 (dedicated sequencing in batches of 10)
• Cost breakdown:
  • DNA extraction: \$5
  • Panel enrichment: \$55 x panel size/100
  • Sequencing: \$1-\$53
• Capex: Thermocycler (\$10-20k), TapeStation (\$6-30k), Nanodrop (\$15k), ONT GridION sequencer (\$50k) or MiSeq i100 (\$100k)

Experimental Modules

1. DNA extraction (2h30, 40’ hands-on)
2. Panel enrichment PCR (2h15, 30’ hands-on)
3. Sequencing run (8h-72h depending on the sequencer, 30’ hands-on)

Ops & Throughput

Turnaround: 2 days (day 1 extraction, day 2 library prep + sequencing)

Hands-on time: 2h30

Parallelizability: High. All steps can be done in parallel for as many samples as needed.

Bottlenecks: Tapestation (16 lanes) and thermocycler (96 wells).

Batching: 1 to 16 samples per technician.

Automation readiness: Full, with commercial solutions available.

Outsourceability: Yes.

Data scale: 100k reads/sample, <1Gb/sample

Data API

Raw format: FASTQ (via POD5 for ONT) Processed format: Variant Call Format (VCF) Resolution: gene level mutation

Analysis Ecosystem

1. QC and cleaning
   • fastqc: Quality control of the run
   • cutadapt: Trimming of sequencing adapters from the reads
2. Alignement:
   • bowtie2
   • minimap2
3. Variant calling
   • Sniffles2
   • PAV
   • svim
   • pbsv

Public datasets

• Genotype-Tissue Expression (GTEx): RNAseq from all major organs from a subset of individuals.
• Gene Expression Omnibus (GEO): Repository of sequencing data from publications
• European Nucleotide Archive (ENA): Repository of sequencing data from publications

Pitfalls & Failure Modes

• Panels with few genes (<20) or highly related genes will have low sequence complexity (all fragments will have similar sequences), which will lead to bad sequencing performance on sequencing-by-synthesis sequencers. To avoid this issue always sequence those amplicons with a complex library (e.g phiX or RNAseq).
• High molecular weight DNA (20-50kb) is fragile and cannot be extracted like low molecular weight DNA. Harsh mechanical manipulations like forcing through porous medium or pipetting too harshly lead to strand breakage. TODO The recommended method is trizol extraction which is cheap but requires good cleaning of the DNA.
• High molecular weight DNA in water is very viscuous. Don’t hesitate do add more buffer to enable manipulation or start with less cells. Always pipette very slowly to avoid breaking the strands. If your solution because less viscuous after pipetting up and down repeatedly it’s likely than you broke the strands.

Related publications

• Tracking of ALK mutations in the blood of lung cancer and neuroblastoma patients: Horn2020, Angeles2021, Heeke2025

Order list

Short amplicon panel (sequenced at \$300/Gb on small short read sequencer) Note that the cheapest single sequencing kit on the market as of September 2025 is the MiSeq i100 Series 5M Reagent Kit (300 cycles) which can accomodate 10-50 panels in parallel. Whenever you can try to sequence panels on runs with more high throughput samples to save about \$50 per panel. Panels barely take any reads which means they won’t affect your complexity or your output significantly.

Oxford nanopore for long amplicon panels. We assume 20x multiplexing.

Protocol variations

• For small panels (<10 genes), you will get a faster turnout and cheaper costs with Sanger Sequencing (e.g \$10/sample with Eurofins)
• Optimized panels are commercially available for many human genes involved in diseases (e.g Ion AmpliSeq)
• There are two main ways to enrich a DNA sequence. “Amplification” uses PCR to specifically amplify the sequence of interest. “Capture” fragments the DNA an captures the fragments containing the sequence of interest with biotinylated oligos and streptavidin-coated beads and sequences the enriched fraction. Amplification is limited to about 30kb with long-range PCR while capture is in theory not limited in size. Capture also provides a bit more context around the target sequence.
• Whole Exome Sequencing is a variation of capture-based panel sequencing with a panel consisting of >400k exonic sequences.
• Adaptive sampling is an amplification-free approach available on ONT sequencing platforms where only strands with features of interest are sequenced.

_{^{This post is part of a series on the cost of experiments. All costs are orders of magnitude and are susceptible to have changed between the post and your order date. All costs assume you perform the whole pipeline in house and do not include labor costs. For outsourcing a decent first estimate is to double the indicated costs. Cheap consumables are not always included if they affect less than 1% of the cost. Always check the protocols coming with the kits for the complete list of consumables to order.}}