Choosing the sequencing approach and the checks around it: why a 16s rrna sequencing service answers composition rather than function and what the region choice decides about resolution, where bacterial whole genome sequencing is the better answer and what it costs in assembly and annotation, what dna genotyping delivers when the variants are known in advance, how ngs library quantification and ngs adapters decide whether a pooled run is balanced, and what sequencing service providers and a dna sequencing service should be asked about deliverables, contamination controls and turnaround before samples are shipped

Scoping the work

1. Choose amplicon or genome from the question. Composition across many samples suits a marker gene survey. Strain level resolution, function and antimicrobial genes need whole genome sequencing. Deciding after sampling is the expensive route.
2. Name the region for an amplicon survey. Different variable regions give different apparent composition and different resolution, and results across regions are not comparable. Fix the region and report it with every result.
3. Run contamination controls in low biomass work. Reagents and kits carry their own bacterial DNA, which dominates low biomass samples. Extraction blanks and no template controls carried all the way through sequencing are essential rather than optional.
4. Quantify libraries properly before pooling. A pool balanced on a rough concentration gives wildly uneven read counts. Quantification by amplification or by an appropriate fluorometric method, plus a size check, is what makes a pool even.
5. Check adapter and index compatibility. Index collisions and adapter mismatches waste a run. Confirm the index set is compatible with the instrument's chemistry and with any other libraries sharing the flow cell.
6. Agree deliverables and controls in writing. Raw reads, the pipeline with versions, the reference database and its version, per sample quality metrics, and the blanks sequenced alongside. A composition table alone cannot be re-analysed.

The database is part of the result

Taxonomic assignment depends on the reference database and its version, and the same reads assigned against two databases produce different tables. That is a methodological choice, not a detail.

Report the database and version with the composition, and keep the reads so the assignment can be redone when the database improves.

Low biomass is a different discipline

Below a certain input, reagent contamination and index hopping contribute a substantial share of the reads. Studies in that regime need blanks, dedicated reagent lots and a sceptical analysis.

Decide early whether your samples are in that regime, because the controls have to be designed in rather than added later.

Common questions

Does a marker gene survey tell you what the community does?

No. It tells you what is there, at a resolution set by the region chosen. Function requires whole genome or transcriptome sequencing, and inferring it from composition is an estimate rather than a measurement.

Why sequence blanks?

Because extraction kits and reagents carry bacterial DNA that can dominate a low biomass sample. Without blanks carried through the whole process, contaminant taxa are indistinguishable from real ones.

Why are my read counts so uneven?

Library quantification before pooling, usually. Rough concentration measurements do not account for adapter ligation efficiency or fragment size, and the pool ends up unbalanced.

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