RCA Sequencing

Description of Service

We accept RCA (rolling circle amplification) products by popular demand from customers, but make no guarantee of the quality of results. RCA samples must be < 25 kb.

Cytiva makes a kit used by some of our customers.

Important notes:
  • RCA tends to make chimeric DNA, which can cause errors in our assembly algorithms.
  • RCA samples are not eligible for reruns because failures are typically due to inadequate sample quality.

This service is performed using the newest long-read sequencing technology from Oxford Nanopore Technologies , and includes the following components:

  • Constructing an amplification-free long-read sequencing library using the newest v14 library prep chemistry, including minimal fragmentation of the DNA in a sequence independent-manner.
  • Sequencing the library with a primer-free protocol using the most accurate R10.4.1 flow cells (raw data is delivered in .fastq.gz format).
  • Generating a high-accuracy circular consensus sequence from the raw reads.
  • Generating a set of feature annotations from the consensus sequence.

Data & File Types Delivered

When your results are ready, you will receive an email notification. Once you sign in to your account, you can download these results from your Dashboard. More detailed information about data & file types can be found in the Results Interpretation Guide.

  • Consensus sequence (.fasta file): Polished consensus sequence of the RCA product.
  • Consensus sequence (.gbk file): Polished consensus sequence of the RCA product, with a molecular map and feature annotations.
  • Molecular map (.html file): An interactive version of the molecular map.
  • Read length histogram (.png file): Displays the read length distribution of the raw reads produced by your sample.
  • Virtual gel (.png file): Displays the raw read lengths from all samples in the order in a virtual gel format.
  • Chromatogram (.ab1 file): Displays the relative abundance of each nucleotide (A, T, G, C) for all raw reads that align to the consensus at each position of the sequence.
  • Coverage plot (.png file): Displays the relative sequencing coverage at each position of the consensus sequence.
  • Per-base data (.txt and .tsv files): Includes 3 sub-files for each sample:
    • SAMPLE.tsv: Indicates how well the raw reads agree with the consensus sequence at each position.
    • SAMPLE_multimer_analysis.txt: Indicates the % distribution of the various concatemer forms of the consensus sequence (monomer, dimer, trimer, etc.).
    • SAMPLE_summary.tsv: Indicates the length, average coverage, relative composition (by moles and mass), total reads, total bases, and %. E. coli genomic DNA contamination for the consensus sequence.
  • Raw read sequences (.fastq.gz file): Provides the sequences of individual raw reads that align to the consensus. Please note that these reads are NOT delivered in the default download, but can be downloaded separately by clicking the Download Raw FASTQ button at the top of the Order Information page. Also note that any raw reads that do not align to the consensus (e.g. host genomic DNA, lower abundance molecular species) are excluded.
Our ability to deliver these target outputs is directly dependent on the quantity and quality of the RCA product sent to us, so we do not guarantee results.

Preparing Your RCA Samples

Submit at least 10 μl of each unpurified RCA reaction in 200 μl strip tubes.

Interpreting Your Results

Please refer to our Results Interpretation Guide for details.