TITLE: Next generation shotgun library preparation for Illumina sequencing - low volume
AUTHORS: cweihe , Julio Avelar-Barragan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:center"><span style = "font-weight:bold;">Adapted from: </span><span>https://www.protocols.io/view/low-volume-methodology-for-nextera-dna-flex-librar-be6rjhd6 and </span><a href="https://www.protocols.io/researchers/w2y25413x2y2" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Julio Avelar-Barragan</span></a><span> protocol from Katrine Whiteson's lab</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:center"><span style = "font-weight:bold;">Last modified: </span><span>May 6</span><span style = "vertical-align:super;">th</span><span>, 2021</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div class = "text-block"><span style = "font-weight:bold;">Objective: </span><span>To prepare a DNA sequencing library for Illumina sequencing. </span></div><div class = "text-block">This protocol can be done over two days, with a stopping point after PCR amplification.</div><div class = "text-block">This protocol presents a low volume methodology for the Illumina DNA Prep Kit (96 Samples). This method increases the number of sequencing libraries which can be generated using each kit from 384 samples (4 x 96-wells) to 864 samples (9 x 96-wells).</div></div>

[STEPS]
?. [PREPARATION]
Take your DNA extractions from  and thaw on ice - spin down on bench spinner.
-20 °C
?. [PREPARATION]
Optional: Measure DNA concentration of your DNA extractions with Qubit (see Qubit protocol).
?. [PREPARATION]
Optional: Based on the DNA concentrations adjust the samples to ~equal ng for library prep.The input volume is  of  DNA. The input range  in ng can be    to
5 µl
35 ng
50 ng
The input amount is not as critical hence step 3 is optional and if too much DNA is added it will be washed away in the downstream steps. As low as 2.5 ng input DNA for some samples created a successful library. If low biomass samples are handled initial quantification is advisable to maximize success.
?. [PREPARATION]
Take Bead linked transposons (BLT) and Tagmentation buffer (TB1) out of the fridge to bring them to room temperature for ~.
?. [PREPARATION]
Prepare 300 ug/mL proteinase K
?. [PREPARATION]
Thaw KAPA HiFi 2X Ready Mix on ice, invert to mix and spin on a bench spinner
?. [PREPARATION]
Thaw and dilute Kapa F/R primers to , on ice.You will need  of each per 96-samples.
55 µl
?. [PREPARATION]
Enter tagmentation protocol on thermal cycler (save as TAGflex):
?. [PREPARATION]
Thaw the i5 and i7 indexes on ice. Index stocks should be diluted to  working stocksYou will need of each per reaction
1.25 µl
Index tubes: spin down briefly (do not vortex as they are small DNA fragments)Index plates: spin down at 1500 rpm to ensure no droplets remain on the plate cover
?. [PREPARATION]
Enter tagmentation stop protocol on the thermal cyclers (save as TAGstop)
?. [PREPARATION]
Enter PCR protocol on thermal cycler (save as flexPCR)
?. [PREPARATION]
Enter and save the protK protocol on the thermal cyclers (save as protK) for running the proteinase K reaction.
?. [TAGMENTATION]
Add of DNA of all the samples to a 96 well plate. If less than 5 ul is used add the remaining volume as nuclease free water.
5 µl
?. [TAGMENTATION]
Seal and spin down the plate with the DNA   to ensure DNA is pelleted in the bottom of the wells
Centrifuge: 1000 33
?. [PREPARATION]
1.  for  followed by a hold at 2. Enable the heated lid (to 100°C if the option is available)
55 °C
10 °C
?. [PREPARATION]
1.  for  followed by a hold at 2. Enable the heated lid (to 100°C if the option is available)
37 °C
10 °C
?. [PREPARATION]
a.  forb.  for c. 12 cycles of:i. for ii.  for iii.  for d.  for e. hold
72 °C
98 °C
98 °C
62 °C
72 °C
72 °C
4 °C
?. [PREPARATION]
a.  for b.  for c. hold
37 °C
68 °C
4 °C
?. [TAGMENTATION]
Make the tagmentation master mix (TMM)
?. [TAGMENTATION]
Vortex BLT vigorously before use and ensure the beads are evenly resuspendedTMM contains  BLT and  TB1 per reaction. For 96 samples it is good to use 110 ul BLT and 110 ul TB1.
1 µl
1 µl
Keep  the beads in the TMM mixed at all times
?. [TAGMENTATION]
Pre-load  of TMM per well in an 8-strip of PCR tubes to use for easy aliquoting into the 96-well plate with DNA samples. Make sure TMM is well mixed before aliquoting and distributing it into the wells with the DNA.
27.5 µl
?. [TAGMENTATION]
Add   of TMM to each DNA well using an 8-channel pipette and mix by pipetting gently ten times to fully mix.
2 µl
If beads are adhered to the top or side of the 96-well plate, briefly centrifuge and fully resuspend the bead pellet by pipetting until  thoroughly mixed
?. [TAGMENTATION]
Seal the plate with a PCR plate cover, place in a thermal cycler, and run the TAGflex program.  2. Proceed immediately to the next steps upon completion of the TAGflex cycle
?. [POST TAGMENTATION CLEAN UP]
While waiting for the TAGflex program, preload  of Tagmentation stop buffer (TSB) into each tube of a 8 strip PCR tube for easy aliquoting.
15 µl
?. [POST TAGMENTATION CLEAN UP]
Gently remove the plate seal from 96-well plate and add TSB into each tagmentation reaction using an 8-channel pipette       Mix by gently pipetting the entire volume, ensuring the beads are fully resuspend
1 µl
?. [POST TAGMENTATION CLEAN UP]
Seal the plate, place in a thermal cycler, and run the TAGstop program
?. [POST TAGMENTATION CLEAN UP]
Remove plate seal and place the plate on a 96-well plate magnet for  or until solution is clear
?. [POST TAGMENTATION CLEAN UP]
Carefully remove the supernatant using an 8-channel pipette without disturbing the beads, change tips between rows
The following 2 steps (24 and 25 1.) can be done sequentially by row  while on the magnet to ensure that the beads are not drying out. This is in particular useful if you have a lot of samples.
?. [POST TAGMENTATION CLEAN UP]
Load  (for 96 reactions) Tagmentation wash buffer (TWB) into a sterile reservoir to pipette from
3.5 mL
?. [POST TAGMENTATION CLEAN UP]
Add  TWB to each well using an 8-channel pipette. 2. Remove the plate from the magnetic stand . Mix by gently pipetting (slowly, to minimize foaming) until beads are        fully resuspended, pipette up and down at least 10 times3. Ensure  beads are submerged in TWB at all times
20 µl
More TWB might be needed to ensure that the beads will be submerged.
?. [POST TAGMENTATION CLEAN UP]
Place the plate on the magnetic stand for or until solution is clear
?. [POST TAGMENTATION CLEAN UP]
Repeat steps 24-26 for a total of two washes and finally resuspend the bead pellet in TWB. Pipet at least 10x until beads are fully resuspended. Seal the plate and keep on the magnet until step 30. Make sure beads are submerged in TWB otherwise add more.
20 µl
?. [AMPLIFY TAGMENTED DNA]
Prepare PCR master mix on ice. Per reaction (i.e. in case of 96 samples calculate for 120) you will need: nuclease free water Kapa F primer Kapa R primer Kapa Hifi Ready Mix
2.75 µl
0.5 µl
0.5 µl
6.25 µl
?. [AMPLIFY TAGMENTED DNA]
For 96 samples pre-load  of PCR master mix into  8 strip PCR tubes.
150 µl
?. [AMPLIFY TAGMENTED DNA]
The following steps can be done row by row to avoid excessive drying of beads and easier resuspension. 1.Remove the TWB wash buffer from the samples from step 27 while on the magnet. 2. Immediately add  of PCR master mix to each well to avoid drying out the beads 3. After all rows received master mix,  take the plate off the magnet and  mix by pipetting up and down until beads are fully resuspended.
10 µl
?. [AMPLIFY TAGMENTED DNA]
Add  of  i5 and i7 barcodes each. Set a P10 to 10 ul and pipette to mix. Then seal the plate and spin down briefly.
Each well will receive a different barcode combination of i5 and i7. Both indexes will be added in equimolar amounts to each well as a total of 2.5 ul (i5= 1.25 ul + i7 =1.25 ul for each well). It is recommended to design a plate map to keep track of the barcode combinations and to ensure that each well (sample) has a unique barcodes combination!
1.25 µl
?. [AMPLIFY TAGMENTED DNA]
Transfer PCR plate into PCR thermocycler and run the "flexPCR" program.
?. [AMPLIFY TAGMENTED DNA]
While the PCR is running pre-load 8 strip PCR tubes with of  proteinase K
35 µl
?. [AMPLIFY TAGMENTED DNA]
Remove the PCR plate from the thermal cycler and spin down at  for  to ensure that all the liquid is on the bottom of the wells.
Centrifuge: 1000 33
?. [AMPLIFY TAGMENTED DNA]
The following 4 steps (33, 35-37) are not required if you directly continue to step 39. Add  of the proteinase K  to each well (step 34). Mix by pipetting 10 ul volume 10 x. Seal the plate and spin briefly.
2.5 µl
?. [AMPLIFY TAGMENTED DNA]
Transfer PCR plate to thermal cycler and run the "protK" program.
?. [AMPLIFY TAGMENTED DNA]
If stopping at this point seal the plate with sealing foil for PCR plates and store at  for up to .
4 °C
?. [POST PCR LIBRARY CLEANUP]
Place the plate on the magnet for or until supernatant is clear. Transfer supernatant into new 96 well plate, avoid the beads.
?. [POST PCR LIBRARY CLEANUP]
If equal DNA amounts were started with you can go ahead and take  of each sample and pool into a 1.5 ml microcentrifuge tube. Otherwise, quantify each sample using qubit or picogreen to pool equal amounts of DNA here.
2 µl
?. [POST PCR LIBRARY CLEANUP]
Vortex and spin down the pool briefly.
?. [POST PCR LIBRARY CLEANUP]
Transfer  of the pool to another 1.5 ml microcentrifuge tube, recommended is a low binding microcentrifuge tube.Store the remaining pool at in case the size selection needs to be optimized.
45 µl
-20 °C
?. [POST PCR LIBRARY CLEANUP]
Vortex and invert SPB multiple times to ensure full resuspension. To aim for an average fragment size of 300 bp follow the protocol below. Prepare SPB-MM (mastemix) by mixing  of SPB with  of nuclease free water. Mix and invert SPB-MM several times.
56 µl
29 µl
The SPB amount in step 43 and step 49 are relevant for the size selection of the library. If the size of the library is not as hoped, for these steps can be repeated with another aliquot of the pool and different amounts of SPB.
?. [POST PCR LIBRARY CLEANUP]
Add  of SPB-MM to of the pool from step 43. Mix thoroughly by pipetting up and down several times. This is a critical step for proper size distribution for the library.
85 µl
45 µl
?. [POST PCR LIBRARY CLEANUP]
Prepare fresh 80% ethanol.Thaw Resuspension buffer and bring to RT and vortex.Equilibrate the sample purification beads (SPB) at RT for then vortex and invert several timesIf the plate was stored spin down the plate for  at  to make sure all the liquid is on the bottom of the well.
Centrifuge: 1000 33
?. [POST PCR LIBRARY CLEANUP]
Incubate at room temperature for
?. [POST PCR LIBRARY CLEANUP]
Place the microcentrifuge tube on a magnetic stand for  or until the supernatant is clear
?. [POST PCR LIBRARY CLEANUP]
Transfer  supernatant into a new 1.5mL tube without disturbing the beads
125 µl
?. [POST PCR LIBRARY CLEANUP]
Vortex and invert the SPB (undiluted stock) until thoroughly resuspended
?. [POST PCR LIBRARY CLEANUP]
Add  SPB (undiluted stock) to the new 1.5mL microcentrifuge tube containing the supernatant
12 µl
?. [POST PCR LIBRARY CLEANUP]
Pipette  volume up and down ten times to thoroughly mix
120 µl
?. [POST PCR LIBRARY CLEANUP]
Incubate at room temperature for .
?. [POST PCR LIBRARY CLEANUP]
Place the microcentrifuge tube on a magnetic stand for . or until the supernatant is clear
?. [POST PCR LIBRARY CLEANUP]
Warm up the elution buffer RSB at  to increase final library yield
50 °C
?. [POST PCR LIBRARY CLEANUP]
Remove and discard the supernatant from step 52 without disturbing the beads
?. [POST PCR LIBRARY CLEANUP]
With the tube on the magnetic stand, add  ethanol without mixing and incubate for Add enough  ethanol so that the beads are entirely submerged
200 µl
?. [POST PCR LIBRARY CLEANUP]
Pipette to remove the ethanol without disturbing the beads
?. [POST PCR LIBRARY CLEANUP]
Repeat steps 55 - 56 one more time for a total of 2 washes
?. [POST PCR LIBRARY CLEANUP]
Carefully, remove any excess liquid from the tube using a pipette without disturbing the beads, try your best to remove all liquid!
?. [POST PCR LIBRARY CLEANUP]
Air-dry beads on the magnetic stand for ~3-5 min.
?. [POST PCR LIBRARY CLEANUP]
Remove the 1.5mL tube from the magnetic stand and add of warm RSB to the beads
32 µl
?. [POST PCR LIBRARY CLEANUP]
Pipette mix until thoroughly resuspended at least 15 times
?. [POST PCR LIBRARY CLEANUP]
Incubate at room temperature on bench for
?. [POST PCR LIBRARY CLEANUP]
Place the 1.5mL tube back on the magnetic stand for  or until clear
?. [POST PCR LIBRARY CLEANUP]
Transfer  supernatant into a new 1.5 ml microcentrifuge tube. Make sure no beads get transferred along with the supernatant.
30 µl
?. [POST PCR LIBRARY CLEANUP]
A subsample can now be submitted for QC: Quantification by qubit and to see the size distribution running the pool on the Bioanalyzer. If the size distribution is not as hoped for go back to step 42 using some of the remaining pool and adjust the amounts of SPB.