Short answer: it’ll sequence just fine on the iSeq 100, but classic V3–V4 (~∼460 bp; e.g., 341F/805R) will not overlap with 2×150 reads, so you can’t merge pairs. That limits many standard 16S pipelines unless you adapt your analysis.

Here’s the practical breakdown:

Read length vs amplicon size

V3–V4 is ~420–470 bp depending on primers.

iSeq 100 is fixed at 2×150 → 300 bp total; you’ll have ~120–170 bp gap between R1 and R2. No overlap → no contig.

What that means for analysis

Pipelines expecting overlapping pairs (e.g., DADA2 paired-end merge) will fail.

Two viable paths:

1. Single-end only: Use just R1 (or just R2) at 140–150 nt after quality trimming. Train your classifier on trimmed lengths (e.g., QIIME 2 sklearn classifier trained on the same region/length). You’ll get decent genus-level calls; species-level will be rarer.

2. Non-overlapping paired strategy: Some workflows allow denoising R1 and R2 separately and classifying each read independently, then reconciling at the taxon level. (Still no contig; think of it as two short independent tags from the same insert.)

If you want overlapping pairs

Switch to MiSeq 2×250 (v2 kit) or 2×300 (v3 kit). That’s the usual setup for V3–V4.

Or change targets to V4-only (~253 bp; 515F/806R), which works great on 2×150 with comfortable overlap.

Library prep with Illumina overhangs

Your locus-specific primers with Illumina overhangs (the standard “tailed” primers) are fine. Do the second PCR to add indices/adapters as usual.

Low diversity considerations (very important on iSeq/MiniSeq)

16S is low-complexity. Spike in 10–20% PhiX to stabilize color balance and improve quality metrics.

If your primers include heterogeneity spacers, you can sometimes reduce PhiX, but most labs still add it.

Throughput / multiplexing sanity check

iSeq 100 yields roughly ~3.8–4.0 million paired reads per run.

If you aim for ≥25–50k reads per sample (single-end or paired but unmerged), you can multiplex on the order of 75–150 samples per run after allowing for PhiX and uneven pooling.

QC tips

Trim adapters/primers; set truncation lengths so your single-end read preserves high Q-scores (often 140–145 nt).

Train/tune your taxonomic classifier on the same region and read length you actually keep.

Include mock communities and negative controls to check for contamination and classifier performance.

Bottom line

Feasible? Yes.

Best practice on iSeq 2×150? Either (a) switch to V4-only so reads overlap, or (b) run V3–V4 as single-end (use R1) and accept modestly lower taxonomic resolution.

Need full V3–V4 with merged pairs? Use a MiSeq 2×250/300 run instead.

Not recommended. The 16S V3–V4 amplicon (≈460 bp; e.g., 341F-805R) requires paired-end overlap to assemble high-quality contigs. iSeq 100 maxes at 2×150 bp (≈300 bp total), leaving a ~160 bp gap—insufficient for merging after quality trimming—thus impairing chimera detection and taxonomic resolution. For iSeq, target a shorter region (e.g., V4 only, ~250 bp) or move the V3–V4 workflow to a MiSeq 2×250/2×300 run to ensure ≥20–50 bp overlap and robust downstream analyses.