TITLE: Long Mate Pair Library Construction Protocol
AUTHORS: Graham J Etherington, Darren Heavens, David Baker, Ashleigh Lister, Rose McNelly, Gonzalo Garcia, Bernardo Clavijo, Iain Macaulay, Wilfried Haerty, Federica Di Palma

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Long Mate Pair Library Construction Protocol</div></div>

[STEPS]
?. For the Tagmentation reactions  and  of Genomic DNA was prepared in  and then  5x Tagment Buffer Mate Pair (Illumina) added followed by  Mate Pair Tagmentation Enzyme (Illumina) and the reaction gently vortexed to mix.
3 µg
3 µg
308 µl
80 µl
12 µl
?. This was then incubated for  at , 100μl of Neutralize Tagment Buffer (Illumina) added and then incubated at  for .
55 °C
0 Room temperature
?. A 1x volume bead clean-up was performed with CleanPCR beads and the DNA eluted in  of Nuclease free Water .
165 µl
?. A  aliquot was run on a BioAnalyser 1200 chip and DNA concentration determined using a Qubit HS Assay.
1 µl
?. Strand Displacement was performed by combining  of tagmented DNA,  10x Strand Displacement Buffer (Illumina),  dNTPs (Illumina) and  Strand Displacement Polymerase (Illumina).
162 µl
20 µl
8 µl
10 µl
?. This was then incubated at  for .
0 Room temperature
?. A 0.75x volume bead clean-up was performed with CleanPCR beads and the DNA eluted in  of Nuclease free Water and the eluted DNA from the  and  reactions pooled.
16 µl
3 µg
3 µg
?. A  aliquot was diluted 1:6 and run on a BioAnalyser 1200 chip and DNA concentration determined using a Qubit HS Assay.
1 µl
?. Size selection was performed on a Sage Science ELF (Sage Science, Beverly, USA). The  in each of collection wells was replaced with fresh buffer and the collection and elution current checked prior to loading the sample. To  of the pooled Strand Displaced reaction  of loading solution was added and then loaded onto a 0.75% Cassette which was configured to separate the sample for  and then each fraction eluted for .
30 µl
30 µl
10 µl
?. Post size selection, the  from each of the 12 collection wells was recovered and the DNA concentration determined using a Qubit HS Assay.
30 µl
?. Circularisation was performed by combining  of size fractionated DNA,  of 10x circularisation buffer (Illumina),  Circularisation Enzyme (Illumina) and nuclease free water.
30 µl
12.5 µl
3 µl
85 µl
?. These were then incubated at  overnight. Linear DNA was digested by adding  Exonuclease (Illumina) and incubation at  for  was followed by  for  to denature the enzyme and  of stop ligation (Illumina) added.
30 °C
3.75 µl
37 °C
70 °C
5 µl
?. During exonuclease treatment  of M280 Dynabeads (Thermo Fisher) were prepared by washing twice with  Bead Bind Buffer (Illumina) before resuspending in  Bead Bind Buffer.
240 µl
600 µl
1560 µl
?. Circularised DNA was then sheared in a  volume on a Covaris S2 for 2 cycles of  with a duty cycle of 10%, cycles per burst of 200 and intensity of 4.
130 µl
?. To  fragmented DNA  of washed M280 beads was added, mixed and then placed on a lab rotator at  for .
130 µl
130 µl
0 Room temperature
?. Library molecules bound to M280 beads were then washed four times with  Bead Washer Buffer (Illumina) and twice with  Resuspension Buffer (Illumina).
200 µl
200 µl
?. A master mix containing  nuclease free water,  10x End Repair Reaction Buffer (NEB, Hitchin, UK) and  end repair enzyme mix (NEB) was prepared and  added to each tube, mixed with the beads and incubated at  for .
1105 µl
130 µl
65 µl
100 µl
0 Room temperature
?. End repaired library molecules bound to M280 beads were then washed four times with  Bead Washer Buffer and twice using  Resuspension Buffer.
200 µl
200 µl
?. A master mix containing  nuclease free water,  A Tailing 10x Reaction Buffer (NEB) and  A tailing enzyme mix (NEB) was prepared and  added to each tube, mixed with the beads and incubated at  for .
325 µl
39 µl
26 µl
30 µl
37 °C
?. To the A tailed library molecules  of the appropriate Illumina Index adapter (Illumina) was added and mixed then  of Blunt/ TA ligase (NEB) added and incubated at  for .
1 µl
31 µl
37 Room temperature
?. Post incubation  of stop ligation added  and then the adapter ligated library molecules bound to M280 beads were then washed four times with  Bead Washer Buffer and twice with  Resuspension Buffer.
5 µl
200 µl
200 µl
?. A master mix containing  nuclease free water,  2x Kappa HiFi (Kappa Biosystems) and  Illumina Primer Cocktail (Illumina) was prepared and  added to each tube, mixed with the beads and the contents, including beads, transferred to a  PCR tube.
240 µl
300 µl
60 µl
50 µl
200 µl
?. Each sample was then subjected to amplification on a Veriti Thermal Cycler (Thermo Fisher) with the following conditions:  for , 8, 10 or 12 cycles of PCR depending upon copy number entering circularisation of  for ,  for ,  for  followed by  for  and Hold at .
98 °C
98 °C
60 °C
72 °C
72 °C
4 °C
?. Post amplification the PCR tubes were placed on a magnetic plate, the beads allowed to pellet and then  of the PCR transferred to a  Lobind Eppendorf Tube. To this  beads of CleanPCR beads were added to precipitate the DNA, the beads washed twice with 70% ethanol and the final library eluted in  resuspension buffer.
45 µl
2 ml
31.5 µl
20 µl
?. Library QC was performed by running a  aliquot on a High Sensitivity BioAnalyser chip and the DNA concentration measured using the High Sensitivity Qubit.
1 µl
?. Libraries to be sequence were then pooled based on DNA concentration and the quantification of the pool was determined by the Kappa qPCR Illumina quantification kit with the pool run at 10pM on a HiSeq with a 2x250bp reads read metric.
?. Reads generated were then processed through NextClip which takes LMP FASTA reads and looks to categorise them into four groups based on the presence of the Nextera adapter junction sequence. Category A pairs contain the adaptor in both reads, Category B pairs contain the adaptor in only read 2, Category C pairs contain the adaptor in only read 1, Category D pairs do not contain the adaptor in either read. NextClip also uses a k-mer-based approach to estimate the PCR duplication rate while reads are examined.