Atto-Seq™

SeqLL’s unique approach to…

RNA Sequencing (RNA-Seq)

Atto-Seq, SeqLL’s unique version of amplification free RNA Seq, is a service generating RNA Sequencing information. Our capabilities take RNA sequencing a step farther, allowing researchers to completely avoid ligation and up front PCR. Thus, the known biases associated with those techniques are avoided and reproducibility and performance are improved.

Highlights:

  • Accuracy and molecule counting
  • No amplification
  • Degraded material specialist and small sample size required
  • First strand cDNA can be fragmented or sequenced DGE-style.
  • Highly reproducible data results, there is excellent linear correlation over 5-4 log dynamic range
  • Read lengths of 33-100nt’s
  • 20-30 million usable reads per channel
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RNAseq Data Analysis

Atto-Seq™ - RNA Seq - sequences cDNA copies of RNA

SeqLL, LLC, has expanded upon the Helicos Single Molecule Sequencing (SMS) platform to deliver the best platform for those seeking accuracy with cDNA based RNA Seq. SMS represents the shortest path between the native RNA population and the final measurement providing the most accurate reflection of that population. Simultaneously, sensitivity of detection is guaranteed by the unbiased representation of virtually every molecule from the starting sample in the final dataset. The SMS technology works to prevent bias and loss of data, achieving accuracy in molecule counting, by requiring fewer steps in Atto-Seq™ - RNA Seq sample preparation than do other approaches in the Sequencing industry. While others use PCR amplification, ligation, and sample size selection, SeqLL based technology does not. The SeqLL approach is very direct - Ribodepleted RNA is converted to cDNA, which is then tailed, blocked, and sequenced. SeqLL provides the best results in the industry for degraded material, small sample size, and low fold detection rates (detection is under two).

SeqLL’s RNA Sequencing Process

Once the flow cells have been loaded onto the HeliScope for sequencing, a template map is created through taking an initial base image. The fluorescent dye and terminator moieties are then removed. Then, new polymerase and the next single fluorescent nucleotide are added. Lasers illuminate 1100 fields of view and the cameras photograph the fluoresced spots. While one flow cell is going through the fluidics process – cleaving the just photographed dye, rinsing, adding the next fluorescent base, rinsing – the other flow cell is going through the imaging cycle – illuminating with the laser and photographing. This process is repeated 120 times with each step rotating through the nucleotides A, C, T, and G… (ie there will be 30 images of As, 30 images of Cs, 30 images of Ts, and 30 images of Gs for a total of 120). With two flow cells having a total of 50 channels being sequenced; there will be the result of 10-20 million usable reads on average for each sample run. It is important to realize that each read represents measurement of an independent molecule of native RNA (for DRS) or its cDNA copy (for RNA Seq).