Cost of long read RNA sequencing

Why do you do this experiment?

Long-read RNA sequencing enables the identification and quantification of RNA expressed in a cell or a sample (the transcriptome) at the isoform resolution.

Input 300ng polyA+ RNA or 1ug total RNA (~300k cells)

Output Fastq file (20-100M PE reads) -> Gene expression

Strategic Value

Cost & Scale

• Variable per run: \$600/sample \$315 (cDNA) - \$1160 (direct RNA)
• Cost breakdown:
  • RNA extraction: \$56
  • Long-read library preparation: \$50 - \$150
  • Sequencing (5M reads, 60Gb): \$
• Capex: Thermocycler (\$10-20k), TapeStation (\$6-30k), ONT PromethION sequencer (\$50-500k) or PacBio sequencer (\$250k-600k)

Experimental Modules

1. RNA extraction (2h30, 40’ hands-on)
2. Sequencing library preparation (2h15, 30’ hands-on)
3. Sequencing run (48-72h depending on the sequencer)

Ops & Throughput

Turnaround: 3+ days (day 1 extraction, day 2 library prep, day 3 or later sequencing 48-72h)

Hands-on time: 4h

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

Bottlenecks: availability of sequencer (4-16 samples on Revio, 24 on ONT P24) 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: 20-100M reads/sample, ~60Gb/sample

Data API

Raw format: FASTQ (via POD5 for ONT) Processed format: count matrix Resolution: transcript-level expression, single nucleotide variants

Analysis Ecosystem

1. Basecalling (ONT)
   • dorado: Official base caller by ONT
   • remora
2. QC and cleaning
   • fastqc: Quality control of the run
   • cutadapt: Trimming of sequencing adapters from the reads
3. Alignement:
   • minimap2
   • LRA
4. Gene expression quantification:
   • htseq-count: Gene-read overlap counts
   • salmon: Quantification taking into account bias in the sequencing method
5. Differential expression
   • DELongSeq for isoform differential expression.
   • DESeq2 or PyDESeq2
   • edgeR or edgePy
   • Sleuth
6. Variant calling
   • Sniffles2
   • PAV
   • svim
   • pbsv

Public datasets

• Genotype-Tissue Expression (GTEx): Long-read 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

• High molecular weight RNA (>1kb) is fragile and cannot be extracted like low molecular weight RNA. Harsh mechanical manipulations like forcing through porous medium or pipetting too harshly lead to strand breakage. The recommended method is trizol extraction which is cheap but requires good cleaning of the RNA.
• High molecular weight RNA in water is quite viscuous (not as bad as DNA though). 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. See ONT guide for more details.
• Long read RNA sequencing methods relying on cDNA use polyA primers to generate the cDNA so will be exclusively composed of mRNA and lncRNA. If you are interested in other long RNAs (because if you are interested in short ones you should go for cheaper per read short read sequencing) use polyA tailing, eventually after ribo-depletion.

Related publications

• PardoPalacios2024: Systematic assessment of long-read RNA-seq methods for transcript identification and quantification
• Helal2024: Benchmark of long-read aligners
• Sakamoto2019: Overview of the benefits of long-read sequencing for cancer genomics
• Ebbert2019: Uncovering the “dark” genome with long-read sequencing
• Glinos2022 “Transcriptome variation in human tissues revealed by long-read sequencing”
• ONT transcriptome pipeline
• Wang2024: Customizing ONT base-calling to improve detection of modifications

Order list

Oxford nanopore starting from extracted RNA.

Pacific Bioscience starting from extracted RNA.

Protocol variations

• 10X genomics used to provided so-called linked reads sequencing where long reads were isolated in droplets, fragmented, and the fragments barcoded with the same barcode.

_{^{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.}}