Immediate visual feedback is modern technology

Collibri next-generation sequencing (NGS) library protocols include visual feedback at every step of the process, enabling the highest chance of library success. It is no longer acceptable to invest 1–2 days in library preparation only to find out at the end of the process if libraries were generated.

Visual feedback now available for the following:

Tracking dyes help you avoid errors

Now you can correct mixing errors in real time. In the examples shown below, one well of a 96-well plate did not receive sufficient mixing or the appropriate reagent. The well is a different color, giving you a visual cue to pause and add the missing reagent and/or mix, and then continue with library preparation, enabling the highest chance of success.

Whole transcriptome sequencing

Invitrogen Collibri Stranded RNA Library Prep kits for Illumina systems detect differential gene expression and alternative polyadenylation from >98% stranded libraries. Ribosomal removal and library generation are completed in under 6.5 hours and ribosomal removal reagents are available for human, mouse, and rat.

The protocols are suitable for degraded or intact samples.

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Detection of alternative polyadenylation, as demonstrated by the RAN gene in the Alternative Adenylation Data Base (APADB) v2, is seen in data from Collibri Stranded RNA libraries.

Library quantification with reduced hands-on time

The Collibri Library Quantification kits offer improved consistency with less hands-on time compared to Kapa library quantification options.  All components are optimized and ready to use:

  • Single-tube master mix containing:
    • Platinum II Taq Hot Start DNA Polymerase
    • dNTPs
    • Primer mix targeting P5 and P7 adapters
    • SYBR green dye
    • Universal concentration passive reference dye enabling a single protocol for all qPCR instruments (high ROX and low ROX)
    • Inert blue tracking dye for real-time process feedback
  • Sample dilution buffer with yellow tracking dye
  • Six DNA standards with yellow tracking dye
Collibri and KAPA quantification kits demonstrate a high level of correlation bar graph

Collibri and Qubit quantification methods demonstrate a high level of correlation. RNA-seq NGS libraries were generated using 100 ng–1 μg of sample using the manufacturer's standard recommended input from a range of sample types. All libraries were quantified using the Collibri Library Quantification Kit and Qubit assay.

The Invitrogen Qubit dsDNA HS Assay is a fluorometric assay that uses dsDNA-binding dyes in order to accurately determine NGS library concentration, and benefits from a simple workflow of just a few minutes per sample.  While the Qubit method has good accuracy and sensitivity, the system is designed to read samples one at a time, thus the workflow does not scale well above 20–30 samples.

The qPCR-based Collibri method scales well for larger sample batches and is the ideal method for precious samples or clinical samples.  The Qubit assay has slightly lower levels of accuracy, but the quick workflow and included quantitation software make it ideal for routine quantitation of smaller numbers of samples. Regardless of the assay that is chosen, users should use good laboratory technique in order to ensure accurate measurement of library concentrations and high-quality Illumina sequencing data.

  Qubit fluorometer Collibri qPCR
Specificity Fluorophore preferentially binds dsDNA Only library fragments containing P5 and P7 adapters are measured
Accuracy Within 15% Within 10%
Sample input requirements 1–20 µL of diluted sample 4 µL of diluted sample
Hands-on time 5-minute setup;
3 seconds for each sample measurement
30-minute setup
(96-well plate)
Time to process 80 samples 25 minutes 90 minutes

Library amplification

The Platinum SuperFi Library Amplification Master Mix is a ready-to-use solution designed for amplification of NGS libraries. The master mix includes Platinum SuperFi DNA Polymerase in combination with a proprietary reaction buffer containing all necessary components to provide exceptionally strong proofreading activity for high amplification accuracy. The Platinum SuperFi Library Amplification Master Mix is not recommended for amplification of primers that contain uracil.

Platinum SuperFi DNA polymerase provides superior fi delity bar graph

Platinum SuperFi DNA polymerase provides superior fidelity. Polymerase fidelity was measured by NGS. Fragmented E. coli DNA (~300 bp) was amplified using Platinum SuperFi Polymerase and other high-fidelity DNA polymerases. The polymerase error rates (error per base pair per cycle) were calculated using bioinformatics techniques. The polymerase fidelities (1/error rate) were normalized to the fidelity of Taq polymerase, whose fidelity value was set at 1. The background level of experimental errors was estimated from PCR-free library sequencing data and subtracted from error rates.

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