Plasmid/Amplicon-seq – Genomics Shared Resource

Plasmid/Amplicon-seq

This service provides complete sequencing, assembly, and annotation of plasmid and amplicon constructs using Oxford Nanopore long-read technology.

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Run Schedule & Turnaround
Run Days	Monday and Thursday every week
Turnaround Time	1–2 days from submission

Plasmid Submission Requirements
Plasmids <20 Kb	At least 20 ng/µL in 20 µL volume
Plasmids >20 Kb	At least 50 ng/µL in 20 µL volume
Maximum Concentration	Up to 100 ng/µL for all submissions
Buffer	Low-TE buffer or 10 mM Tris-HCl pH 8.0 (such as Qiagen EB or equivalent). EDTA concentrations >5 mM may result in failed sequencing.
Tube Format	1.5 mL tubes with sample numbers written legibly on the top and sides. Data delivery cannot be guaranteed for samples with illegible labels.

Amplicon Submission Requirements
Amplicon Size	500–5,000 bp in length (we are interested in testing outside this range)
Primer Design	Primers should flank amplicons by 15–20 bp, since the beginning and end of the sequence can be of low quality
Concentration	100 ng in 20 µL (5 ng/µL)
Cleanup	PCR reactions should be cleaned and eluted in TE or nuclease-free water, and free of contaminating DNA/RNA sequences
EDTA Limit	Elution buffer should have <5 mM EDTA (standard TE preparations are 1 mM)

Data Delivery
Data are processed using EPI2ME Labs pipelines and delivered to your RaDar account:
Plasmids (wf-clone-validation)	Consensus FASTA – assembled plasmid sequences GenBank (.gbk) – auto-annotated features for viewing in SnapGene or other sequence viewers
Amplicons (wf-amplicon)	Consensus FASTA – assembled amplicon sequences GenBank (.gbk) – auto-annotated features
Raw Reads	FASTQ files are available for download for one month at rcweb.dartmouth.edu/GSR_Active/Plasmid-seq. Directories are named by run date with sub-directories for each sample, named by barcode ID.

Checking Assemblies in SnapGene

You can verify your plasmid/amplicon-seq assemblies by aligning the raw FASTQ reads to your reference plasmid map in SnapGene:

1. Open your reference plasmid sequence in SnapGene (it must be circular).
2. Go to Tools > Align to Reference Sequence.
3. Drag and drop the FASTQ file from the raw reads folder into the alignment dialog, or use "Browse" to select it.
4. Click Align to generate the alignment.
5. In Map View, aligned reads appear as arrows above the reference. Switch to Sequence View to inspect individual base calls and identify any mismatches or indels.

For a detailed walkthrough, see SnapGene's guide: Align a Whole Plasmid Sequence to a Reference

Sample Dropbox Locations

Genomics and Molecular Biology Core – Rubin 6, Bench 62, black freezer under bench

Remsen Shared Resource Lab – Remsen 241 (collected at 7 am daily)

Borwell Loading Dock – DHMC

Computer Science and Engineering (Thayer) – Enter from student offices (ECSC 134), first freezer on right. Please email GMBSR@groups.dartmouth.edu if dropping samples at Thayer.

Note: The plasmid/amplicon-seq workflow is robust to a range of plasmid sizes and sequence features. Plasmid preps with significant contamination (more than one plasmid present) and those with highly repetitive content may prove challenging to assemble. Assemblies may also present with single nucleotide gaps not present in the reference sequence. Assembly quality can be confirmed by aligning raw reads to the consensus sequence in SnapGene (see above).