FAQ: Is Monarch-purified gDNA compatible with Next Generation Sequencing (NGS) platforms like PacBio and nanopore?

Yes. gDNA purified with this kit can be used for various applications on these sequencing platforms. Assuming high quality input material has been used, read lengths of up to 50 kb can be routinely achieved using Monarch purified gDNA from different starting materials.

Monarch purified DNA quality is superior to SDS Proteinase K-purified DNA commonly used for Nanopore sequencing

2 µg E. coli genomic DNA extracted using either Monarch or by following a traditional SDS Proteinase K isopropanol protocol was enzymatically fragmented with identical incubation times and used as input DNA for MinION library preparation. Oxford Nanopore Technology’s 1D kit protocol was followed to prepare libraries. Both libraries were run on the MinION, the data collected for 48 hours and base called using Albacore. Monarch-purified DNA performed better on all sequencing metrics, including read length, read quality and total sequencing data collected. The graphs illustrate that Monarch purified data provides a higher percentage of high quality sequencing data than SDS Proteinase K purified DNA.

Monarch provides excellent quality starting material for Pacific Biosciences (PacBio) sequencing

Bacterial gDNA was isolated from 5x109 E. coli ER2683 cells with pACYC184 plasmid by following the Monarch rapid protocol for Gram- bacteria. 10 µg Monarch-purified gDNA was sheared with a Covaris® g-TUBE, targeting 10 kb fragment size. 5 µg sheared DNA was used for library preparation according to the standard PacBio protocol (without size selection). Samples were sequenced on a Pacific Biosciences RSII sequencer, using the P6/C4 chemistry, loaded as 10 pM SMRTbell library and data were collected in a 5 hr movie.

Total bases (yield): 1208 Mb, Average Polymerase read length: 16,046, Polymerase read quality: 0.85. Average Reads of Insert length: 6,060, Reads of Insert quality: 0.89, Longest read of insert: 43,680.

A. Insert lengths indicate that the majority of the reads were in the desired insert size range. 
B. The polymerase read lengths indicate that the purified DNA was of high quality to allow the enzyme to read the insert several times within the SMRTbell construct. 
C. The loading evaluation data further illustrates that the purified DNA is of high quality.