Pyrosequencing, RNA-seq, and Long Reads

Pyrosequencing

Pyrosequencing is sequencing by synthesis read with light ¹. Nucleotides are dispensed one type at a time in a known order. When the polymerase incorporates one, it releases pyrophosphate, which ATP sulfurylase turns into ATP, which luciferase uses to make a flash of light; apyrase then clears leftover nucleotide before the next dispensation ¹.

Reading a pyrogram:

• A peak at a dispensation means that base was added; no peak, not added.
• Peak height is proportional to the number of identical bases added - a double-height G means GG ¹. Long homopolymers get less accurate.
• At a position with two alleles, the two peaks split the height, giving a quantitative percentage.

That quantitation is pyrosequencing’s niche: percent methylation at each CpG after bisulfite conversion (MGMT in glioblastoma), and mutant percentage at hotspots (KRAS, BRAF), sensitive to roughly 5%. Reads are short (tens of bases), and it’s been displaced in many labs by NGS, but remains a reliable targeted tool.

RNA-seq

RNA-seq sequences cDNA made from RNA, so it measures what genes are expressed and how much (by counting reads per gene), plus splice variants and fusion transcripts ¹.

• Library prep starts with RNA: remove ribosomal RNA (or select poly(A) mRNA), fragment, reverse-transcribe, then build a normal library.
• Clinically, targeted RNA panels detect gene fusions in solid tumors and leukemias (ALK, ROS1, RET, NTRK, BCR::ABL1) more reliably than DNA, because introns, where DNA breakpoints scatter, are spliced out.
• Expression signatures classify some cancers and predict recurrence risk.

RNA quality matters: degraded FFPE RNA is assessed by DV200 before library prep (see Assessing Quality and Quantity).

Long reads

Long-read (third-generation) platforms sequence single molecules without clonal amplification, producing reads of thousands to hundreds of thousands of bases ¹:

Why it matters:

• Repeat expansions: a read longer than the repeat sizes it exactly (FMR1, C9orf72).
• Structural variants, inversions, and complex rearrangements.
• Phasing: whether two variants are on the same copy (cis) or opposite copies (trans), critical for compound heterozygosity in recessive disease.
• Methylation read directly from the native DNA, without bisulfite.
• Difficult regions: pseudogenes (CYP2D6, SMN1/SMN2) and highly repetitive DNA.

Per-base accuracy and cost have improved enough that long reads are moving into clinical genomes.

What to take from this

Pyrosequencing dispenses one nucleotide at a time and reads light from released pyrophosphate; peak heights count bases and give quantitative methylation or mutant percentages. RNA-seq sequences cDNA for expression and, clinically, fusion detection. Long-read single-molecule platforms span repeats and structural variants, phase variants, and read methylation directly.