Authors: Janet Hill, Bonnie Chaban, Jennifer Town, Matthew Links & Tim Dumonceaux
This protocol describes an alternative approach to performing Roche’s Paired End Rapid Library Preparation Method for 8 kb span libraries. This method uses the Corvaris g-tube for DNA fragmentation, eliminating the need for a HydroShear apparatus, and a Sage Science BluePippin electrophoresis platform to size select the 8 kb fragments, eliminating the need for agarose gel electrophoresis and sample recovery using a Whatman Elutrap device. These two modifications allow the protocol to be completed in significantly less time (eliminate an overnight step) and require significantly less capital investment in laboratory equipment. This modified library preparation protocol has yielded paired end libraries meeting all the quality control benchmarks the original protocol stipulates and has been combined with sequencing on a GS Junior instrument to generate excellent sequencing results. The equipment substitutions presented allow paired end library preparation to be practical for smaller research groups.
In large-scale sequencing centers it is commonplace to have a range of DNA sequencing platforms (e.g. 454, Illumina, SOLiD, and PacBio) and also specialized devices for shearing of DNA molecules common in the preparation of paired-end libraries. With the introduction of laboratory-scale devices like the GS Jr and MiSeq there is a shift occurring where DNA sequencing technology is becoming directly accessible within small labs. The ability to perform whole genome sequencing and assembly within a lab is now reasonable to consider for Bacteria using the GS Jr. A crucial type of data for scaffolding of genome assemblies is paired-end data. By generating information from sequencing reads linked across some known or estimated spanning distance, bioinformatic techniques are capable of ordering and orienting contigs produced from shotgun sequencing. In terms of the GS Jr platform the system is inherently capable of sequencing paired-end libraries but there is a challenge for laboratory-scale use as the conventional protocol for library production includes shearing of DNA molecules using a Hydroshear. As a specialized device the Hydroshear represents a significant investment for a laboratory-scale setting and may present a barrier to the use of paired-end sequencing in lab settings. This protocol describes an alternative approach to performing Roche’s Paired End Rapid Library Preparation Method for 8 kb span libraries. This method uses the Corvaris g-tube for DNA fragmentation, eliminating the need for a HydroShear apparatus, and a Sage Science BluePippin electrophoresis platform to size select the 8 kb fragments, eliminating the need for agarose gel electrophoresis and sample recovery using a Whatman Elutrap device. These two modifications allow the protocol to be completed in significantly less time (by eliminating an overnight step) and simplify sheared fragment purification. This modified library preparation protocol yielded paired-end libraries meeting all the quality control benchmarks of the original protocol and has been combined with shotgun sequencing on a GS Junior to generate excellent assemblies for a range of Bacteria. The equipment substitutions and associated savings in time provide a practical protocol for laboratory-scale paired-end sequencing on the GS Jr.
Reagents unique to modified protocol:
Regents in common with original Roche paired end rapid library preparation method:
Equipment unique to modified protocol:
Equipment in common with original Roche paired end rapid library preparation method:
Base protocol is the Paired End Rapid Library Preparation Method Manual, 20 kb and 8 kb Span, GS Junior Titanium Series, March 2012. See attached Supplementary Manual.
Modifications by Hill lab (Saskatoon) group:
Start with 15 µg of sample genomic DNA in 150 µL Tris-HCl pH 8.0 (genomic DNA preparation of 100 ng/µL) – same as original protocol
Section 3.1 – DNA Fragmentation (HydroShear) – completely disregard this section. Instead, use Covaris g-tubes.
Section 3.2 – Fragment End Repair
Section 3.3 – Circularization Adaptor Ligation
Section 3.5 – Fill-In Reaction
Section 3.6 – Circularization
Section 3.7 – Nebulizer Assembly (3.7.1) and DNA Nebulization and Collection/Purification of the Fragmented DNA (3.7.2)
Section 3.8 – Fragment End Repair
Section 3.9 – Immobilization Bead Preparation
Section 3.10 – Adaptor Ligation
Section 3.11 – Library Immobilization
Section 3.12 – Library Amplification
Section 3.13 – Sizing Mix Preparation
Section 3.14 – Final Library Size Selection
Section 3.15 – Library Quality Assessment
Sections 3.1 to 3.5 can be performed in a day (including the BluePippin run). A good spot to pause overnight is either right after the BluePippin run (end of Section 3.4) or after the fill-in reaction and quantification (end of Section 3.5). Sections 3.5 or 3.6 to the end of Section 3.11 is a good second day of work. Finishing Section 3.12 to 3.15 on the third morning allows for plenty of time to continue on with sequencing emPCR later in that day.
Things that we have noted:
Figure 1 shows typical results of shearing with the Covaris g-tubes and the purification of appropriately sized fragments using the Blue Pippin. Table 1 shows sequencing metrics generated on the GS Junior apparatus for three bacteria.
Figure 1: Gel electrophoresis of sheared DNA
Table 1.: Sequencing metrics. Sequencing metrics using shotgun and paired-end protocols on the GS Junior (Titanium chemistry). Results are shown for three genomes: Pseudomonas sp. BRG100, Brachyspira sp. KL180, and Pantoea agglomerans isolate 4.
Paired End Rapid Library Preparation Method Manual, 20 kb and 8 kb Span, GS Junior Titanium Series, March 2012
Source: Protocol Exchange. Originally published online 26 August 2014.