| 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.
| Run Schedule & Turnaround | |
| Run Days | Monday and Thursday every week |
| Turnaround Time | 1–2 days from submission |
| General Submission Requirements | |
| 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. |
| EDTA Limit | Elution buffer should have <5 mM EDTA (standard TE preparations are 1 mM) |
| 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. |
| 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 |
| 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 |
| 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 Result summary (.html) – a plain-language report for every sample: whether it assembled cleanly, assembled only after short-read rescue, or could not be completed, including the expected vs. assembled size and recommended next steps |
| Amplicons (wf-amplicon) |
Consensus FASTQ – assembled amplicon sequences with per-base quality scores AB1 (.ab1) – synthetic Sanger-style chromatogram derived from the consensus, analogous to traditional Sanger output. Can be uploaded to tools that require AB1 input, such as Synthego ICE for CRISPR knockout/knockin scoring. Result summary (.html) – a plain-language report for every sample: reads received, read length, consensus length and quality, and coverage depth, or the likely cause and next steps if a consensus could not be generated |
| Raw Reads | A single combined FASTQ file per sample is available for download for one month at rcweb.dartmouth.edu/GSR_Active/Plasmid-seq. Each run date contains one sub-folder per sample (named by sample ID), each holding a single combined .fastq.gz. |
| Checking Assemblies in SnapGene |
| You can verify your plasmid/amplicon-seq results by aligning the raw FASTQ reads to your reference sequence in SnapGene:
1. Open your reference sequence in SnapGene (a plasmid must be circular). 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).