TITLE: Diatom DNA library preparation for Illumina Miseq Sequencing using the rbcL marker gene 
AUTHORS: Cecile Chardon, Valentin Vasselon, Marine Vautier, Sonia Lacroix, Agnès Bouchez, Frédéric Rimet, Isabelle Domaizon

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is part of the DNA workflow applied in the Eco-ALpsWater Project, here in particular to characterize the diversity of diatom assemblage in biofilms or plankton samples.</div><div class = "text-block"><span>Different studies have already revealed the potential of diatom metabarcoding applications for freshwater quality assessment (Kermarrec et al. 2014; Vasselon et al. 2017ab; Visco et al. 2015). The choice of the marker gene and barcode region is key for obtaining relevant inventories of diversity and precise taxonomic assignment. For benthic diatoms, the </span><span style = "font-style:italic;">rbc</span><span>L gene has proved to be an appropriate taxonomic marker for biomonitoring (Kermarrec et al. 2013, 2014; Vasselon et al. 2017a,b) and a well‐curated barcode reference library is already available to assign species names to </span><span style = "font-style:italic;">rbc</span><span>L sequences (R‐Syst::diatom, Rimet et al. 2016).</span></div><div class = "text-block">For the Eco-AlpsWater project, biolfilms sampled in rivers and lakeshores are collected as described in the dedicated protocols (“Lake plankton sample collection ...” and “Biofilms sample collection ...”) and DNA is extracted as described in the protocol “Biofilms DNA extraction” ; all these protocols are part of the Deliverable D.T1.1.2. We present here the following step in the DNA workflow (i.e. PCR amplification of selected barcodes, and wet lab methods to prepare DNA library for downstream MiSeq Sequencing). This protocol has been used in recent studies (e.g. Keck et al 2018 ; Vasselon et al 2018) where diatoms metabarcoding has been used for ecological assessment of rivers.</div><div class = "text-block"><span>Several primers were proposed in the literature to characterize Diatom communities through environmental DNA metabarcoding approaches, including the 18S, COI and </span><span style = "font-style:italic;">rbc</span><span>L barcodes. Following the recommendation provided by Kermarrec et al. 2014, who compared the efficiency of those 3 barcodes to accurately characterize diatom communities from freshwater samples (lakes and rivers), the </span><span style = "font-style:italic;">rbc</span><span>L barcode will be used within the Eco-AlpsWater project as he provides a good taxonomical resolution.</span></div></div>

[STEPS]
?. [Primers preparation]
At the start of a new DNA library preparation, you must prepare "fresh" primers at 10 µM, the volume required to process all the samples and make aliquots to avoid freezing / thawing of primers.
?. [Primers preparation]
Global informationsThe original diatom rbcL primers include the Diat_rbcL_708F as forward primer (5’ AGG TGA AGT TAA AGG TTCATA CTT DAA 3’, Stoof-Leichsenring et al. 2012) and the R3 as reverse primer (5’ CCT TCT AAT TTA CCA ACA ACT G 3’, Bruder et al. 2007) for an expected amplicon length of 312 bp.An updated version of those primers was proposed in Vasselon et al. 2017 and will be used within the Eco-AlpsWater project. The forward and the reverse rbcL primers are respectively composed by an equimolar mixiture of 3 foward rbcL primers (Diat_rbcL_708F_1, Diat_rbcL_708F_2, Diat_rbcL_708F_3)  and 2 reverve rbcL primers (R3_1, R3_2).              Type of   primer   Name   Sequence      Forward_1   Diat_rbcL_708F_1   AGGTGAAGTAAAAGGTTCWTACTTAAA      Forward_2   Diat_rbcL_708F_2   AGGTGAAGTTAAAGGTTCWTAYTTAAA      Forward_3   Diat_rbcL_708F_3   AGGTGAAACTAAAGGTTCWTACTTAAA      Reverse_1   R3_1   CCTTCTAATTTACCWACWACTG      Reverse_2   R3_2   CCTTCTAATTTACCWACAACAG     Several protocols can be proposed to perform HTS library preparation, which may vary according to the strategyATA CTT DAA 3’, Stoof-Leichsenring et al. 2012) and the R3 as reverse primer (5’ CCT TCT AAT TTA CCA ACA ACT G 3’, Bruder et al. 2007) for an expected amplicon length of 312 bp.An updated version of those primers was proposed in Vasselon et al. 2017 and will be used within the Eco-AlpsWater project. The forward and the reverse rbcL primers are respectively composed by an equimolar mixiture of 3 foward rbcL primers (Diat_rbcL_708F_1, Diat_rbcL_708F_2, Diat_rbcL_708F_3)  and 2 reverve rbcL primers (R3_1, R3_2).   ABC1Type of
  primerNameSequence2Forward_1Diat_rbcL_708F_1AGGTGAAGTAAAAGGTTCWTACTTAAA3Forward_2Diat_rbcL_708F_2AGGTGAAGTTAAAGGTTCWTAYTTAAA4Forward_3Diat_rbcL_708F_3AGGTGAAACTAAAGGTTCWTACTTAAA5Reverse_1R3_1CCTTCTAATTTACCWACWACTG6Reverse_2R3_2CCTTCTAATTTACCWACAACAGapplied to add index on PCR products allowing to multiplex several samples within one HTS run (tagged PCR, ligation,…) , the sequencing technology used (PGM, MiSeq,…) and the sequencing platform requirements.Within the EcoAlps-Water project, a two steps PCR will be applied in order to add dual-index to each individual samples following the sequencing platform requirement for MiSeq Illumina sequencing.For the first PCR, we use the equimolar mixes of Diat_rbcL_708F_1_P5, Diat_rbcL_708F_2_P5, Diat_rbcL_708F_3_P5 and R3_1_P7, R3_2_P7 as forward and reverse primers respectively. Half of the P5 (5' CTTTCCCTACACGACGCTCTTCCGATCT 3') and P7 (5' GGAGTTCAGACGTGTGCTCTTCCGATCT 3') Illumina adapters are included to the 5’ part of the rbcL forward and reverse primers respectively. Those region will serve as primer fixation site for the second PCR  ABC1Type of
  primerNameSequence2Forward_1Diat_rbcL_708F_1_P5CTTTCCCTACACGACGCTCTTCCGATCTAGGTGAAGTAAAAGGTTCWTACTTAAA3Forward_2Diat_rbcL_708F_2_P5CTTTCCCTACACGACGCTCTTCCGATCTAGGTGAAGTTAAAGGTTCWTAYTTAAA4Forward_3Diat_rbcL_708F_3_P5CTTTCCCTACACGACGCTCTTCCGATCTAGGTGAAACTAAAGGTTCWTACTTAAA5Reverse_1R3_1_P7GGAGTTCAGACGTGTGCTCTTCCGATCTCCTTCTAATTTACCWACWACTG6Reverse_2R3_2_P7GGAGTTCAGACGTGTGCTCTTCCGATCTCCTTCTAATTTACCWACAACAG
ABC1Type of
  primerNameSequence2Forward_1Diat_rbcL_708F_1AGGTGAAGTAAAAGGTTCWTACTTAAA3Forward_2Diat_rbcL_708F_2AGGTGAAGTTAAAGGTTCWTAYTTAAA4Forward_3Diat_rbcL_708F_3AGGTGAAACTAAAGGTTCWTACTTAAA5Reverse_1R3_1CCTTCTAATTTACCWACWACTG6Reverse_2R3_2CCTTCTAATTTACCWACAACAG
ABC1Type of
  primerNameSequence2Forward_1Diat_rbcL_708F_1_P5CTTTCCCTACACGACGCTCTTCCGATCTAGGTGAAGTAAAAGGTTCWTACTTAAA3Forward_2Diat_rbcL_708F_2_P5CTTTCCCTACACGACGCTCTTCCGATCTAGGTGAAGTTAAAGGTTCWTAYTTAAA4Forward_3Diat_rbcL_708F_3_P5CTTTCCCTACACGACGCTCTTCCGATCTAGGTGAAACTAAAGGTTCWTACTTAAA5Reverse_1R3_1_P7GGAGTTCAGACGTGTGCTCTTCCGATCTCCTTCTAATTTACCWACWACTG6Reverse_2R3_2_P7GGAGTTCAGACGTGTGCTCTTCCGATCTCCTTCTAATTTACCWACAACAG
?. [Primers preparation]
PreparationEquimolar mixture of rbcL primers at 100µM  -    for foward rbcL primers : mix 1volume of Diat_rbcL_708F_1_P5 (at 100µM), 1volume of Diat_rbcL_708F_2_P5 (at 100µM) and 1volume of Diat_rbcL_708F_3_P5 (at 100µM)-    for reverse rbcL primers : mix 1volume of R3_1_P7 (at 100µM), 1volume of R3_2_P7 (at 100µM)Note : ensure that the final volume of the equimolar mix at 100 µM allows to prepare the necessary volume of equimolar mixture at 10 µM1/10 th dilution to have equimolar mixture of rbcL primers at 10µM  -    for foward rbcL primers : mix 1volume of Diat_rbcL_708F_1_P5 (at 100µM), 1volume of Diat_rbcL_708F_2_P5 (at 100µM) and 1volume of Diat_rbcL_708F_3_P5 (at 100µM)-    for reverse rbcL primers : mix 1volume of R3_1_P7 (at 100µM), 1volume of R3_2_P7 (at 100µM)Note : ensure that the final volume of the equimolar mix at 100 µM allows to prepare the necessary volume of equimolar mixture at 10 µM-    for both mix : add 1volume of equimolar mix of rbcL primers at 100µM and 9volume of molecular grade waterNotes : -    ensure that the final volume of the equimolar mix at 10 µM allows to amplify all the samples-    make aliquots to avoid freezing / thawing of primers-    stored aliquots at -20°C
?. [PCR 1]
Important informations : -    Each DNA extracts are amplified in triplicate-    PCR1 amplifications are performed using 1 µL of extracted DNA (max [25ng/µL]) in a final volume of 25μL following mixture and reaction conditions as follow
?. [PCR 1]
Under the specific DNA-work station, prepare one PCR mixture for all samples to be treated, see table belowNotes : -    include in the PCR mixture calculations, 1 negative control, 1 positive control, 1 security sample-    before using a reagent or sample, mix it and make a brief centrifugation-    use refrigerated racks to keep reagents and PCR mixture in cool conditions-    add Taq DNA Polymerase at the last moment, pipetting slowly beacause the Taq DNA Polymerase solution is viscous             Reagents   [Initial]   [Final]   Volume per sample (µL)      Molecular   grade water   -   -   15.6      Buffer   10 X   1 X   2.5      dNTP   2.5 mM   0.2 mM   2      BSA   10 mg/mL   0.5 mg/mL   1.25      Primer F   10 µM   0.5 µM   1.25      Primer R   10 µM   0.5 µM   1.25      TaKaRa   LA Taq® DNA Polymerase   5 U/µL   0.75 U   0.15     Vortex slowly the PCR mixture, make a brief centrifugationDistribute the PCR mixture, in strips of 8 PCR tubes, 24µL per PCR tubeNote :-    You can use the same tip, to distribute the mixture in few PCR tubesAdd samples : 1 µL of extracted DNA (max [25ng/µL])Notes : -    for negative control, add 1µL of solution without diatoms (e.g. molecular grade water)-    for positive control, add 1µL of DNA extract which must be amplified  (e.g. 1µL of DNA extracted from diatom culture or 1µL of DNA extracted from environmental sample which be amplified during a previous PCR)-    volume of DNA  used per PCR reaction can be adjusted if DNA concentration is low and water volume adjusted in consequence (e.g.  if PCR performed with 3µL of DNA, water volume will be set to 13.6 µL); if the concentration of extracted DNA is > 25ng/µL, make a dilution to have a final concentration at 25ng/µL and for the PCR add 1µl of diluted DNAClose strips of 8 PCR tubesVortex slowly the mixture of PCR mixture / DNA, make a brief centrifugationNotes : -    include in the PCR mixture calculations, 1 negative control, 1 positive control, 1 security sample-    before using a reagent or sample, mix it and make a brief centrifugation-    use refrigerated racks to keep reagents and PCR mixture in cool conditions-    add Taq DNA Polymerase at the last moment, pipetting slowly beacause the Taq DNA Polymerase solution is viscous  ABCD1Reagents[Initial][Final]Volume per sample (µL)2Molecular grade water--15.63Buffer10 X1 X2.54dNTP2.5 mM0.2 mM25BSA10 mg/mL0.5 mg/mL1.256Primer F10 µM0.5 µM1.257Primer R10 µM0.5 µM1.258TaKaRa LA Taq® DNA Polymerase5 U/µL0.75 U0.15Vortex slowly the PCR mixture, make a brief centrifugationDistribute the PCR mixture, in strips of 8 PCR tubes, 24µL per PCR tubeNote :-    You can use the same tip, to distribute the mixture in few PCR tubesAdd samples : 1 µL of extracted DNA (max [25ng/µL])Notes : -    for negative control, add 1µL of solution without diatoms (e.g. molecular grade water)-    for positive control, add 1µL of DNA extract which must be amplified  (e.g. 1µL of DNA extracted from diatom culture or 1µL of DNA extracted from environmental sample which be amplified during a previous PCR)-    volume of DNA  used per PCR reaction can be adjusted if DNA concentration is low and water volume adjusted in consequence (e.g.  if PCR performed with 3µL of DNA, water volume will be set to 13.6 µL); if the concentration of extracted DNA is > 25ng/µL, make a dilution to have a final concentration at 25ng/µL and for the PCR add 1µl of diluted DNAClose strips of 8 PCR tubesVortex slowly the mixture of PCR mixture / DNA, make a brief centrifugationNote :-    You can use the same tip, to distribute the mixture in few PCR tubesAdd samples : 1 µL of extracted DNA (max [25ng/µL])Notes : -    for negative control, add 1µL of solution without diatoms (e.g. molecular grade water)-    for positive control, add 1µL of DNA extract which must be amplified  (e.g. 1µL of DNA extracted from diatom culture or 1µL of DNA extracted from environmental sample which be amplified during a previous PCR)-    volume of DNA  used per PCR reaction can be adjusted if DNA concentration is low and water volume adjusted in consequence (e.g.  if PCR performed with 3µL of DNA, water volume will be set to 13.6 µL); if the concentration of extracted DNA is > 25ng/µL, make a dilution to have a final concentration at 25ng/µL and for the PCR add 1µl of diluted DNAClose strips of 8 PCR tubesVortex slowly the mixture of PCR mixture / DNA, make a brief centrifugationNotes : -    for negative control, add 1µL of solution without diatoms (e.g. molecular grade water)-    for positive control, add 1µL of DNA extract which must be amplified  (e.g. 1µL of DNA extracted from diatom culture or 1µL of DNA extracted from environmental sample which be amplified during a previous PCR)-    volume of DNA  used per PCR reaction can be adjusted if DNA concentration is low and water volume adjusted in consequence (e.g.  if PCR performed with 3µL of DNA, water volume will be set to 13.6 µL); if the concentration of extracted DNA is > 25ng/µL, make a dilution to have a final concentration at 25ng/µL and for the PCR add 1µl of diluted DNAClose strips of 8 PCR tubesVortex slowly the mixture of PCR mixture / DNA, make a brief centrifugation
ABCD1Reagents[Initial][Final]Volume per sample (µL)2Molecular grade water--15.63Buffer10 X1 X2.54dNTP2.5 mM0.2 mM25BSA10 mg/mL0.5 mg/mL1.256Primer F10 µM0.5 µM1.257Primer R10 µM0.5 µM1.258TaKaRa LA Taq® DNA Polymerase5 U/µL0.75 U0.15
?. [PCR 1]
Place strips of 8 PCR tubes in thermal cyclerStart the program below  ABC1TemperatureTimeCycles295 °C5 minX1395 °C1 minX33454 °C1 min572 °C1 min672 °C5 minX1710 °Cpause  Note : cycle number can be increased to a maximum of 40 cycles but must be avoided to limit aspecific amplification and to limit biases linked to PCR amplification.
ABC1TemperatureTimeCycles295 °C5 minX1395 °C1 minX33454 °C1 min572 °C1 min672 °C5 minX1710 °Cpause
?. [PCR validation, PCR1 product pool preparation, plate preparation and shipment]
Important informations : Reminders : -    Each DNA extracts are amplified in triplicate-    Final PCR volume is 25μLGet-PlaGe facility requirements : -   Volume final of PCR1 product needed is 50µL and concentration minimum needed is 30ng/µL -   PCR1 products must be placed in 96 wells plates sealed by adhesive film-   PCR1 products must be one after the other in column, without empty wellsRecommandations : -    Each DNA extracts are amplified in triplicate-    Final PCR volume is 25μLGet-PlaGe facility requirements : -   Volume final of PCR1 product needed is 50µL and concentration minimum needed is 30ng/µL -   PCR1 products must be placed in 96 wells plates sealed by adhesive film-   PCR1 products must be one after the other in column, without empty wellsRecommandations : -   Volume final of PCR1 product needed is 50µL and concentration minimum needed is 30ng/µL -   PCR1 products must be placed in 96 wells plates sealed by adhesive film-   PCR1 products must be one after the other in column, without empty wells-  To  limit the risk of contaminations, work in a room dedicated to handling amplicons with dedicated materials (pipettes, specific DNA-work station,...)-    You need to reduce at maximum  freezing / thawing of PCR1 productswe therefore recommend that you :1) carry out the PCR reaction, validate the PCR and prepare the PCR pool over a week and during this period store the PCR1 products at + 4 ° C (in the fridge)2) place the PCR1 product pools at -20°C while you process the other samples3) thawing the PCR1 product pools to prepare the 96 willsplates 2-3 days before plates shipment4) realize shipment in cold conditions (0-4°C), in 1 - 2 days
?. [PCR validation, PCR1 product pool preparation, plate preparation and shipment]
PCR validationNotes : -    details of this protocol are not describe here-    to realize a quality control of PCR product (lenght of fragment and quantity), you can use electrophoresis on agarose gel or other techniques (e.g : TapeStation (Agilent Technologies, Santa Clara, California))-    use maximum of 4µL of PCR product per PCR reaction-    for negative control, no amplification-    for positive control, expected amplicon length ≈ 376 bp-    for samples, expected amplicon length ≈ 376 bp-    for one sample, amplification identical between triplicate of PCR productIf these 4 points are validated, you can pool triplicate of PCR product, one pool per sample
?. [PCR validation, PCR1 product pool preparation, plate preparation and shipment]
PCR1 product pools preparationNotes : -   One pool per sample with a final volume at 57µL-   For one sample, use same tip-  To reduce the risk of errors, write the name of sample and "rbcL product" on the tubeClean a specific DNA - work station (dedicated to handling amplicons) and apply the UV for 15minAnnotate the 1.5 or 2mL tubesVortex slowly the PCR1 products, make a brief centrifugationTake 19µL of PCR1 product  per replicate and place its in corresponding annotated tube (so in total you have 3 X 19µL = 57µL)Store PCR1 product pools (if the plate will be prepared under 2-3 days, store at +4°C otherwise store at -20°C)(so in total you have 3 X 19µL = 57µL)Note : we recommand you to realize and sequence negative control pools. e.g if you have 9 sets of PCR, you can realize 3 negative control pools. The 1st with negative controls of PCR sets 1, 2, 3; The 2nd with negative controls of PCR sets 4, 5, 6; The 2rd with negative controls of PCR sets 7, 8, 9.
?. [PCR validation, PCR1 product pool preparation, plate preparation and shipment]
Plate preparationBefore plate preparation, you need :-    prepare map(s) of plate(s)-    clean a specific DNA - work station (dedicated to handling amplicons) and apply the UV for 15min-    taw the PCR1 product pools-    vortex them slowly and make a brief centrifugation-    place the PCR1 product pools in a rack, in the right orderUnder specific DNA - work station, on 96 wells plate (use reference recommanded by Get-PlaGe facility) write the plate namePipetting 52µL of PCR1 product pool and put them in the right wellChange the tip, and start again with the second sampleRepeat this last step for all PCR1 product poolsNotes : to limit risk of errors, -    fill in the plate per column-    use one new tips box per plate and use tips by column too-    move or discard the PCR1 product pool tube after each useWhen your 96 wells plate is completed, sealed it correctly with adhesive films for PCR recommanded by Get-PlaGe facilityPlace 96 wells plate in a bag and stored at + 4°C (maximum 2-3 days) before the shipment -    prepare map(s) of plate(s)-    clean a specific DNA - work station (dedicated to handling amplicons) and apply the UV for 15min-    taw the PCR1 product pools-    vortex them slowly and make a brief centrifugation-    place the PCR1 product pools in a rack, in the right orderUnder specific DNA - work station, on 96 wells plate (use reference recommanded by Get-PlaGe facility) write the plate namePipetting 52µL of PCR1 product pool and put them in the right wellChange the tip, and start again with the second sampleRepeat this last step for all PCR1 product poolsNotes : to limit risk of errors, -    fill in the plate per column-    use one new tips box per plate and use tips by column too-    move or discard the PCR1 product pool tube after each useWhen your 96 wells plate is completed, sealed it correctly with adhesive films for PCR recommanded by Get-PlaGe facilityPlace 96 wells plate in a bag and stored at + 4°C (maximum 2-3 days) before the shipment Notes : to limit risk of errors, -    fill in the plate per column-    use one new tips box per plate and use tips by column too-    move or discard the PCR1 product pool tube after each useWhen your 96 wells plate is completed, sealed it correctly with adhesive films for PCR recommanded by Get-PlaGe facilityPlace 96 wells plate in a bag and stored at + 4°C (maximum 2-3 days) before the shipment
?. [PCR validation, PCR1 product pool preparation, plate preparation and shipment]
ShipmentPlace bag countaining sealed plate in a box.This box must contain everything necessary to ensure a temperature between 0°C  and + 4°CThe shipment must be made within 1 to 2 daysNote :-    it is preferable to send the box before WednesdaySend an email to the platform with the Excel document required for sample processingNote :-    it is preferable to send the box before Wednesday