This is the GBS method we have optimised based on Elshire 2011, Poland 2012 and Petersen 2012, with golay barcodes from Caporaso 2012. We ususally run 300 samples per lane and get good results from a variety of different plant species.
Currently all the fwd barcodes are the same length so we still need to spike in 20% genomic DNA to keep the sequencer happy when it gets to the cut-site, future modifications will add a second plate of forward adaptors with additional barcode bases to remove this requirement.
Recipe for 5x Annealing Buffer (50mM Tris, 250mM NaCl, 5mM EDTA) to make 50ml:
1M TRIS-Hcl ph8
2.5ml
0.5M EDTA
500µl
1M NaCl
25ml
make up to 50ml with clean H2O
Refer to XXX for the adaptors to order, we have 96 forward adators based on Poland 2012 with 12bp Golay barcodes from Caporaso 2012, and then use 12 reverse adaptors based on Petersen 2012 that allow indexing, with variable length MTC codes to improve the quality of the reverse sequencing read.
To make 40uM of each adaptor add 40ul Fwd adapter oligo, 40ul Rev adapter oligo and 20ul of 5x annealing buffer.
PCR program GBS barcode anneal - incubate at 97.5oC for 2.5 minutes, and then cool at a rate of not greater than 3oC per minute until the solution reaches a temperature of 21oC. Hold at 4oC
Dilute down the adaptors - Fwd adaptors diluted to 5ng/µl (10x) then 0.5ng/µl (working dilution). Rev adaptors kept at 40µM
Normalise DNA
Prepare 100-1000ng DNA in 40µl. Normalise using pico green. If there is variation in the integrity (degradation) pool by integrity when pooling for the pippin prep so that the normalisation of pools evens it out. If you don't do this you may get 2x reads for the high integrity samples. We don't normalise again during the protocol so it is important to get it right at the start.
Add 4µl of P1 barcode adaptor (0.5ng/µl) and 1µl of P2 adaptor to each sample with multichannel pipette and filtered tips taking great care not to mix up the adaptors - this is a critical point where any mistakes will result in "accidental introgression"
Using multichannel pool all 96 samples into one disposable reservoir, mix using P1000 pipette.
Keep 2 x 1.5ml as a safety stop (freeze for later just in case).
Add 500µl of pooled GBS, 500µl of 4M GuHCL and 500ul of 100% EtoH to each of six eppies.
Mix, add contents of all six eppies into one spin column per plate (750µl at a time). Spin down for 1 min and repeat until all GBS has been bound to the silica columns.
Proceed to one wash with 750µl wash buffer, add buffer and then spin for 1 min, discard eluate and then spin for 2 mins to dry column.
Add 100µl of 10mM Tris to each column - incubate 1 min then spin into collection eppie.
Take 40µl of this and size select 300-400bp in the pippin prep, the other 60µl is kept as a second saftey stop (freeze like the other).
PCR amplification
PCR Recipe:
Ingredient
1 sample
11 samples
Phusion 5x HF
6µl
66µl
10mM DNTP
0.6µl
66µl
Illumina Fwd PE
1.2µl
13.2µl
Rev Index primer
1.2µl
13.2µl
Taq
0.24µl
2.64µl
Water
0.76µl
8.36µl
Take 2x 20µl aliquots removed from pippin prep, add 10µl mmix as above for 2 x 30µl reactions per index
PCR machine DDRadAmplify
Note: We usually pool three 96well plates together, and use one index for each, so Index 2, Index 6, and Index 12 as recommended to balance the index reads.
Post PCR Pooling
Check each pool on Shimadzu (5µl sample and 5µl library)
check on Qubit (1µl)
From shimadzu calculate the proportion that is library = library peak/(library peak + other (adaptor) peaks)
Multiply Qubit reading by shimadzu proportion.
Make equimolar pool of indexes using the above figure and including 20% spike-in of genomic shotgun library
Bead Clean
We do one 0.8X bead clean at the end to remove adaptors.
Allow beads to come to room temperature and then vortex them very well to resuspend.
For every 100µl of pooled library add 0.8X = 80µl of beads (ampure or equivalent, we use PCRcleanDX). Mix well by
pipetting.
Incubate on bench for 2-3m.
Place the tube on an appropriate magnetic stand to separate the
beads from the supernatant. If necessary, quickly spin the sample to
collect the liquid from the sides of the tube before placing
on the magnetic stand.
After 5 minutes (or when the solution is clear), carefully remove and
discard the supernatant. Be careful not to disturb the beads that contain
DNA targets (do not discard beads).
Add 200µl of freshly prepared 80% ethanol to the tube while in
the magnetic stand. Incubate at room temperature for 30 seconds, and
then carefully remove and discard the supernatant. Be careful not to
disturb the beads that contain DNA targets.
Repeat the ethanol wash for two washes total. If necessary, briefly spin the tube, place back on the magnet and remove traces of ethanol with a p10
pipette tip.
Air dry the beads for up to 5 minutes while the tube is on the
magnetic stand with the lid open. It is important not to over dry the beads - it can result in lower
recovery of DNA target. Elute the samples when the beads are
still dark brown and glossy looking, but when all visible liquid has
evaporated. When the beads turn lighter brown and start to crack
they are too dry.
Remove the tube from the magnetic stand. Elute the DNA target
from the beads by adding 22µl of 10mM Tris.
Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 minutes at room temperature. If necessary, quickly
spin the sample to collect the liquid from the sides of the tube or plate
wells before placing back on the magnetic stand.
Place the tube on the magnetic stand. After 5 minutes (or when
the solution is clear), transfer 20µl to a new PCR tube, take 1µl of the remaining liquid and check on the Qubit, the pooled library is now ready to ship for sequencing
store at -20oC and ship on blue ice (ice bricks) that have spent the weekend in the -80oC freezer.