{"id":892,"date":"2026-05-01T15:40:23","date_gmt":"2026-05-01T15:40:23","guid":{"rendered":"https:\/\/geiselmed.dartmouth.edu\/gsr\/plasmid-seq\/"},"modified":"2026-05-04T16:59:38","modified_gmt":"2026-05-04T16:59:38","slug":"plasmid-amplicon-seq","status":"publish","type":"page","link":"https:\/\/geiselmed.dartmouth.edu\/gsr\/plasmid-amplicon-seq\/","title":{"rendered":"Plasmid\/Amplicon-seq"},"content":{"rendered":"\n\n\n
Extraction<\/a><\/td>\nNucleic Acid QC<\/a><\/td>\nRNA-seq<\/a><\/td>\nDNA-seq<\/a><\/td>\nSingle Cell<\/a><\/td>\nSpatial<\/a><\/td>\nMicroarray<\/a><\/td>\nSequencing<\/a><\/td>\nPlasmid\/Amplicon-seq<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

This service provides complete sequencing, assembly, and annotation of plasmid and amplicon constructs using Oxford Nanopore long-read technology.<\/p>\n

Place an Order on RaDar<\/a><\/p>\n\n\n\n\n\n
Run Schedule & Turnaround<\/td>\n<\/tr>\n
Run Days<\/td>\nMonday and Thursday every week<\/td>\n<\/tr>\n
Turnaround Time<\/td>\n1\u20132 days from submission<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n\n\n\n\n\n
Plasmid Submission Requirements<\/td>\n<\/tr>\n
Plasmids <20 Kb<\/td>\nAt least 20 ng\/\u00b5L in 20 \u00b5L volume<\/td>\n<\/tr>\n
Plasmids >20 Kb<\/td>\nAt least 50 ng\/\u00b5L in 20 \u00b5L volume<\/td>\n<\/tr>\n
Maximum Concentration<\/td>\nUp to 100 ng\/\u00b5L for all submissions<\/td>\n<\/tr>\n
Buffer<\/td>\nLow-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.<\/td>\n<\/tr>\n
Tube Format<\/td>\n1.5 mL tubes with sample numbers written legibly on the top and sides. Data delivery cannot be guaranteed for samples with illegible labels.<\/strong><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n\n\n\n\n\n
Amplicon Submission Requirements<\/td>\n<\/tr>\n
Amplicon Size<\/td>\n500\u20135,000 bp in length (we are interested in testing outside this range)<\/td>\n<\/tr>\n
Primer Design<\/td>\nPrimers should flank amplicons by 15\u201320 bp, since the beginning and end of the sequence can be of low quality<\/td>\n<\/tr>\n
Concentration<\/td>\n100 ng in 20 \u00b5L (5 ng\/\u00b5L)<\/td>\n<\/tr>\n
Cleanup<\/td>\nPCR reactions should be cleaned and eluted in TE or nuclease-free water, and free of contaminating DNA\/RNA sequences<\/td>\n<\/tr>\n
EDTA Limit<\/td>\nElution buffer should have <5 mM EDTA (standard TE preparations are 1 mM)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n\n\n\n\n
Data Delivery<\/td>\n<\/tr>\n
Data are processed using EPI2ME Labs pipelines and delivered to your RaDar account:<\/td>\n<\/tr>\n
Plasmids
(wf-clone-validation)<\/span><\/td>\n		Consensus FASTA<\/strong> \u2013 assembled plasmid sequences
GenBank (.gbk)<\/strong> \u2013 auto-annotated features for viewing in SnapGene or other sequence viewers<\/td>\n<\/tr>\n
Amplicons
(wf-amplicon)<\/span><\/td>\n		Consensus FASTA<\/strong> \u2013 assembled amplicon sequences
GenBank (.gbk)<\/strong> \u2013 auto-annotated features<\/td>\n<\/tr>\n
Raw Reads<\/td>\nFASTQ files are available for download for one month at rcweb.dartmouth.edu\/GSR_Active\/Plasmid-seq<\/a>. Directories are named by run date with sub-directories for each sample, named by barcode ID.<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n\n
Checking Assemblies in SnapGene<\/td>\n<\/tr>\n
You can verify your plasmid\/amplicon-seq assemblies by aligning the raw FASTQ reads to your reference plasmid map in SnapGene:<\/p>\n

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

For a detailed walkthrough, see SnapGene's guide: Align a Whole Plasmid Sequence to a Reference<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n\n\n\n\n
Sample Dropbox Locations<\/td>\n<\/tr>\n
Genomics and Molecular Biology Core<\/strong> \u2013 Rubin 6, Bench 62, black freezer under bench<\/td>\n<\/tr>\n
Remsen Shared Resource Lab<\/strong> \u2013 Remsen 241 (collected at 7 am daily)<\/td>\n<\/tr>\n
Borwell Loading Dock<\/strong> \u2013 DHMC<\/td>\n<\/tr>\n
Computer Science and Engineering (Thayer)<\/strong> \u2013 Enter from student offices (ECSC 134), first freezer on right. Please email GMBSR@groups.dartmouth.edu<\/a> if dropping samples at Thayer.<\/strong><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Note:<\/strong> 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).<\/p>\n","protected":false},"excerpt":{"rendered":"

Extraction Nucleic Acid QC RNA-seq DNA-seq Single Cell Spatial Microarray Sequencing Plasmid\/Amplicon-seq This service provides complete sequencing, assembly, and annotation of plasmid and amplicon constructs using Oxford Nanopore long-read technology. Place an Order on RaDar Run Schedule & Turnaround Run Days Monday and Thursday every week Turnaround Time 1\u20132 days [\u2026] <\/p>\n

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