Gap Closure

The primer design function within the Gap4 program was used to design primers for gap closure of the Butyrivibrio sp. MB2003 genome project. For regions difficult to sequence, such as ribosomal rRNA operons, repeat regions and regions with possible stable secondary structures, the contigs of interest were visualised using Artemis (Rutherford et al., 2000). BLASTN and BLASTX results for the coding ORFs in that region were examined. The information from the BLAST searches was used to select appropriate regions to allow design of suitable primers. Primers synthesized at 50 nM scale by IDT (Integrated DNA Technologies; http://eu.idtdna.com) were supplied desalted and lysophilized. Primers were reconstituted in sterile dH2O water at a

concentration of 100 µM and stored at -20ºC. PCR reactions and subsequent preparations of PCR products were carried out for all gap closing reactions as described in sections below.

45 Polymerase Chain Reactions

Polymerase Chain Reactions (PCR) were performed in 50 µl volumes using a standard PCR protocol. Constituents were added to dH2O in a 0.2 ml thin wall PCR tube (Quality

Scientific Plastics, Petaluma, CA, USA), in the order listed in Table 2.11, on ice. The solution was mixed using a P200 micropipetter (Thermo Labsystems, Waltham, MA, USA) and a sterile 200 µl pipette tip and subsequently transferred to a PX2 Thermal Cycler (Thermo). The reaction was cycled through a 2 min denaturation at 94ºC, followed by 35 cycles of:

 94ºC for 30 sec

 56ºC for 30 sec

 72ºC for 2 min

The reaction underwent a 2 min elongation at 72ºC and was cooled to 4ºC until examined by gel-electrophoresis.

Table 2.11 PCR assembly of reagents.

PCR constituents Amount per 1x Reaction Final Concentration

10x PCR Buffer, minus Mg 5 µL 1X

10 mM dNTP mixture (each) 4 µL 0.2 mM each

50 mM MgCl2 1.5 µL 1.5 mM

Primer Mix (20 µm each 5'& 3') 0.5 µL each 0.2 µM each

Template DNA (diluted) 1 µL (as required)

Platinum Taq DNA Polymerase 0.2 µL 1.0 unit

dH2O (autoclaved) 40.3 µL N/A

Total Volume 50 µL

Long Range PCR

Long Range PCR was performed using the Eppendorf Triple Master system (Eppendorf, Hamburg, Germany). The reaction was prepared as described in Table 2.12. The reaction was cycled through a 3 min denaturation at 93ºC, followed by 35 cycles of:

 93ºC for 15 sec

 56ºC for 30 sec

 68ºC for 7 min

The reaction was cooled to 4ºC until examined by gel-electrophoresis. Table 2.12 Long Range PCR assembly of reagents.

LR PCR constituents Amount per 1x Reaction Final Concentration

10x HiFi PCR Buffer 5 µL 1X

20 mM dNTP mixture (each) 2 µL 0.2 mM each

50 mM MgSO4 2 µL 2.0 mM

Primer Mix (20 µm each 5'& 3') 1.0 µL each 0.4 µM each

Template DNA (diluted) 1 µL (as required)

HiFi Platinum Taq (5 units/µL) 0.2 µL 1.0 unit

dH2O (autoclaved) 37.8 µL N/A

46 Agarose gel-electrophoresis

Agarose gel-electrophoresis was carried out using Bio-Rad equipment (Bio-Rad, Hercules, CA, USA). Agarose (Invitrogen, Carlsbad, CA, USA) or Low Melt Agarose (Progen, Heidelburg, Germany) was mixed with 1× TAE buffer to give a final concentration of 1.5% w/v agarose or 1% w/v Low Melt Agarose, unless otherwise stated. The mixture was boiled to melt the agarose and then allowed to cool to 50ºC in a waterbath. Gels were cast in a 15× 10 cm casting stand with either a 1.5 mm thick 15 well comb, or a 1.5 mm thick 20 well comb. The gel was allowed to cool to room temperature and solidify. The comb was removed and the gel tray was transferred to a wide Mini-sub® cell GT (Bio-Rad) and immersed in 1× TAE buffer. Unless otherwise stated, 9 µl of each PCR-amplified product was dispensed on to a small square of Parafilm M (American National Can, Chicago, IL, USA), combined with 1 µl of 10× BlueJuiceTM (Invitrogen, Carlsbad, CA, USA) and carefully transferred to a separate well. Also, 8 µl of the 1 Kb+ ladder (Invitrogen, Carlsbad, CA, USA) was added typically to bordering wells. The DNA fragments were separated using a Powerpac Basic applying 80 volts (5 1/3 volts/cm) for 60 min. The gel was stained with 0.5 μg/ml

ethidium bromide for 40 min, then washed in water for 10 min and analysed by UV transillumination at λ=590 nm and photographed using a Gel Logic 200 system (Eastman Kodak Company, Rochester, NY, USA).

PCR clean up

DNA fragments resulting from PCR reactions were purified using a QIAquick® PCR purification kit (Qiagen, Hilden, Germany). Binding (PB) Buffer from the kit (5 volumes) were mixed with the product of each PCR reaction and the solution was applied to a QIAquick spin column and subjected to centrifugation for 60 sec at 13,000×g. During this process, DNA of 0.1 – 10 Kb binds to the column, while all other constituents of the reaction flow through. The flow through was discarded and 750 µl of Elution (PE) Buffer from the kit was applied to the column and centrifuged for 60 sec at 13,000×g. This washing step was repeated once more, discarding the flow–through each time. The column was centrifuged for an additional 1 min at 13,000×g to remove any residual PE buffer, and 30 µl of sterile dH2O was added to the centre of the QIAquick

column and incubated at room temperature for 5 min. The eluate was transferred to a fresh sterile Eppendorf tube by centrifugation at 13,000×g for 60 sec.

47 PCR Product Sequencing

All DNA sequencing reactions of PCR products were conducted by the Allan Wilson Centre Genome Sequencing Service at Massey University, Palmerston North. This service included fluorescent labeling of PCR products using the Big DyeTM Terminator (Version 3.1), a Ready Reaction Cycle Sequencing Kit, subsequent removal of unincorporated fluorescent dideoxy NTPs (ddNTPs) by cleanup and precipitation of products and capillary separation on an ABI3730 Genetic Analyzer (Applied Biosystems Inc., Carlsbad, California). Results were received as ABI tracefiles that were analysed using Trev (Ewing et al., 1998; Bonfield et al., 2002), a DNA trace visualization and editing tool from the Staden package.

Gap Closure

The Butyrivibrio sp. MB2003 ABI trace files received from sequencing of the PCR products were entered into the Gap4 database. The ABI files were pre-processed as summarised in Figure 2.1 and the process were repeated until gaps were closed.

Figure 2.1. Outline of the pre-processing and assembly of the Butyrivibrio sp. MB2003 gap closure sequences.

B. hungateiMB2003 Gap Closure ABI trace files

Trev

Phred

Initialise Experimental Files

Augment Experimental Files

Quality Clip

Assembly entered into Gap4 Database

Primer Design, Scaffolding and Gap Closure

P re ga p 4 G a p 4

Viewer & editor for ABI trace files

Base calling program for DNA sequence trace files. ABI files converted to SCF (Standard Chromatogram File) format (Ewing et al., 1998)

Program converts trace files into experimental (EXP) file format

Added Sanger new naming scheme feature to all EXP files to include information concerning their primers, physical chemistry, templates and the sizes of the inserts used to produce each read

Program removes poor quality sequence

EXP files are entered into the Gap4 database. Find Internal Joins function was run, automatically process the sequences and reports joins between contigs if they exist

R e p e a t a s n e ce ss a ry u n ti l g e n o m e p ro je ct is c o m p le te

48

2.2.15 Annotation of the Butyrivibrio sp. MB2003 genome