Choosing what to sequence and who should do it: why total rna seq and a poly A selected library answer different questions and why the choice cannot be made after the samples are prepared, what rna sequence analysis should hand over besides figures, where ffpe rna seq is limited by the material rather than by the method, how targeted dna sequencing, dna library construction and an ngs liquid biopsy each set their own depth and input requirements, what genomics services, ngs service providers, sequencing service providers, a dna sequencing service and a next generation sequencing machine or ngs instruments quotation are actually pricing, and where a dna sequencing kit, a dna synthesizer and oligosynthesis capability in house change the calculation

Scoping a sequencing project

1. Choose the library fraction from the question. Selection for mature transcripts captures a defined fraction and discards the rest; depletion of ribosomal material keeps non coding and unpolyadenylated species. Degraded material usually forces depletion regardless of preference.
2. Set depth from the effect size, not from a default. Detecting a modest change in an abundant transcript needs far less depth than detecting a rare variant. Work the requirement out statistically before booking, because depth is the main cost lever.
3. Match input requirements to what you have. Every library chemistry has a minimum input and a preferred quality. Precious, degraded or low input samples need chemistries designed for them, and those have their own biases to declare.
4. Agree the deliverables in writing. Raw reads, alignments, the pipeline with versions and parameters, the reference used, per sample quality metrics and the code behind each figure. A report is a summary, not a deliverable.
5. Randomise across preparation batches. Batch, index set and lane all leave signatures. Distribute experimental groups across batches and hand the batch table to whoever analyses the data.
6. Decide in house against a service honestly. Owning a sequencer makes sense at sustained volume with a trained operator; below that, a service gives better data per unit cost and no maintenance. Synthesis capability follows the same logic.

The library is the experiment

Everything downstream is constrained by what the library captured. A question about non coding transcripts cannot be answered from a poly A selected library at any depth, and no amount of analysis recovers what was never sequenced.

Decide the library chemistry with the analyst in the room. It is the decision that most often cannot be undone.

Reproducibility is a deliverable

An analysis that cannot be rerun cannot be corrected or extended. The practical test is whether a competent person, given the handover, can regenerate a named figure without contacting the analyst.

Write that test into the contract. It separates providers who hand over work from providers who hand over pictures.

Common questions

Total or poly A selected?

Selection where mature transcripts are the question and the material is intact; depletion where non coding species matter or the material is degraded. They are not comparable, so mixing them within a study is a confound.

How much depth do I need?

It follows from the effect size and the abundance of what you are measuring. Calculating it beforehand is the difference between an adequately powered study and an expensive underpowered one.

What should a provider hand over?

Raw and aligned data, the exact pipeline with versions, the reference and annotation, per sample quality metrics and runnable code. Anything less cannot be reproduced or extended.

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