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.