Quick Access:
1
Application #1
Evaluation of the
Kapa Hyperprep PCRfree protocol for
the typing of Salmonella clinical
isolates in Magelia
Kapa Hyperprep PCRfree protocol for
the typing of Salmonella clinical
isolates in Magelia
ABSTRACT
Salmonella enterica is the second most reported bacterial cause of food-borne infections in Europe. Therefore, surveillance activities based on pathogen subtyping are an important measure of controlling Salmonellosis by public health agencies. Whole-genome-based typing is becoming the gold standard due to its improved resolution and reduced cost.
In this paper, we performed a comparative blind study for Salmonella enterica typing, comparing side by side standard processing and Magelia processing, Inorevia's multi-omics platform. PCR-free library preparation was used for blind NGS typing of this relevant foodborne pathogen, for samples ranging from 5 ng to 85 ng.
A successful pathogen identification relies on PCRfree approach to avoid any bias that could be introduced by amplification, which in turn requires sufficient DNA amount.
In this paper, we performed a comparative blind study for Salmonella enterica typing, comparing side by side standard processing and Magelia processing, Inorevia's multi-omics platform. PCR-free library preparation was used for blind NGS typing of this relevant foodborne pathogen, for samples ranging from 5 ng to 85 ng.
A successful pathogen identification relies on PCRfree approach to avoid any bias that could be introduced by amplification, which in turn requires sufficient DNA amount.
Average adapter dimer percentages in Manual and Magelia treated samples
In the manually-treated samples, poor quality libraries did not allow for serotype identification due to high levels of background noise for 3 samples out of 8. This would lead the clinician to have to go back and prepare libraries anew for serotype identification, sequence again and analyze the data once more, doubling the cost for these samples.
Conversely Magelia-treated samples exhibited extremely low background noise (adapter dimer %), allowing complete serotype identification, using identical inputs in terms of total DNA as in the manual processing. The high precision magnetic bead handling allowed by Magelia's core technology made this high quality possible on such low inputs.
Conversely Magelia-treated samples exhibited extremely low background noise (adapter dimer %), allowing complete serotype identification, using identical inputs in terms of total DNA as in the manual processing. The high precision magnetic bead handling allowed by Magelia's core technology made this high quality possible on such low inputs.
Mapping for the Panama serovar in the manual and Magelia treated samples in the region of the S. entericagenome conatining SNP c.884A>C
Magelia treatment resulted in better library yield and negligible dimer formation compared to the manual preparation of the eight Salmonella serovars tested. The sequencing analysis clearly demonstrates higher quality sequences obtained and a better coverage achieved when using Magelia, resulting in 100% successful identification of S. enterica serotypes vs 62.5% for standard manual method.
Our multi-omics platform Magelia challenges the minimum required input for a PCR-free library kit, by reducing reagent volumes 8x and DNA input 50x while yielding high quality sequencing libraries.
Our multi-omics platform Magelia challenges the minimum required input for a PCR-free library kit, by reducing reagent volumes 8x and DNA input 50x while yielding high quality sequencing libraries.
Overall Magelia processing of low input samples enabled characterization of clinically relevant pathogens to the single nucleotide resolution:
- Blind identification of all Salmonella serotypes for Magelia treated samples vs. 5/8 samples for manual treatment underline a higher success rate (relevant for regulated environments)
- Optimal cleanup and negligible adapter-dimer formation made possible thanks to our patented magnetic tweezer technology and high precision DNA purification
2
Application #2
Highly efficient RNA depletion for RNA-Seq applications in Magelia
Massively parallel sequencing of RNAs has unlocked the study of the transcriptome, enabling genome wide transcriptional profiling. Whole transcriptomic analysis is hindered by the abundance of ribosomal RNA (rRNA), representing ≥ 80% of molecules in total RNA, making removal of rRNA prior to RNA library prep a crucial step for researcher and clinicians.
In this paper, we have evaluated the advantages of using Magelia rRNA depletion application for Whole RNA-seq. Equal amounts of human control total RNA (100 ng) were treated in parallel manually and in the Magelia for using Illumina’s Ribo-Zero Plus rRNA Depletion Kit. In addition to full automation of the procedure, an average reagent reduction of 2,25-fold was achieved.
In this paper, we have evaluated the advantages of using Magelia rRNA depletion application for Whole RNA-seq. Equal amounts of human control total RNA (100 ng) were treated in parallel manually and in the Magelia for using Illumina’s Ribo-Zero Plus rRNA Depletion Kit. In addition to full automation of the procedure, an average reagent reduction of 2,25-fold was achieved.
As the control RNA used for the study was extracted from a well characterized cell line, the Harmonizome database was used to identify highly transcribed genes across studies. By determining the normalized expression values of the top and bottom 20 transcribed genes reported in the Harmonizome, we found a 7,62- and 8,3-fold difference for the Magelia and manually treated samples respectively, corroborating the biological validity of these data.
More importantly, when we focus on MYC gene mappings, in chromosome 8, we observe an average coverage for this particular region of 210x and 634x for manual and Magelia treated samples respectively. This, once more, highlights improved depth/transcriptome resolution for Magelia treated samples, likely related to improved depletion.
More importantly, when we focus on MYC gene mappings, in chromosome 8, we observe an average coverage for this particular region of 210x and 634x for manual and Magelia treated samples respectively. This, once more, highlights improved depth/transcriptome resolution for Magelia treated samples, likely related to improved depletion.
In conclusions, this unique combination of benefits show Magelia sample preparation effectiveness when using input samples at the kit's lower limit of 100 ng :
- Lower rRNA reads are counted when using Magelia underliying a better rRNA depletion.
- Comparing results to Harmonizome database on expression values of the top and bottom 20 transcribed genes reported underline the validity of obtained data.
- Higher coverage and data resolution are clearly observed, highlighting Magelia core technology efficiency:
- Better reaction kinetics allowing to more efficient enzymatic rRNA removal
- Better magnetic beads manipulation in low volumes
- Reagent volume reduction of 2,25-fold without compromizing on quality data
We will release more applications shortly
Subscribe to our Newsletter to stay tuned on what is coming next!
