COI Metabarcoding

This protocol is designed to amplify the mini COI barcode (313bp) as described in Leray et al (2014). The primer set (mlCOIintF and jgHCO2198) is successfully amplifying a wide range of marine invertebrate phyla. Multiplexing is made possible by a variation of the initial primers tailed on the 5’ end with a 6 bp “tag”.

The protocol described below is tailored for the Illumina MiSeq platform and uses the Illumina Truseq kit. We use single indexing and a hierarchal tagging approach that can be modified depending on your number of samples. The number of samples in each illumina run will depend on the minimum number of sequences needed per sample. This will depend on the expected diversity. As a safety net, we suggest that at least 100,000 sequences be obtained for each sample.

Materials:

1- Primers

The primer sequences are always listed in the 5′ -> 3′ orientation:

mlCOIintF:     GGWACWGGWTGAACWGTWTAYCCYCC (Leray et al, 2014)

jgHCO2198: TAIACYTCIGGRTGICCRAARAAYCA (Geller et al, 2014)

It is also possible to multiplex the samples by using the following tailed primer sets (Table S4 of Leray et al (2015) where the first 6 bp represents the tag.

Primer Label

Primer Sequence (5’ – 3’)

mlCOIint_Tag1

AGACGCGGWACWGGWTGAACWGTWTAYCCYCC

mlCOIint_Tag2

AGTGTAGGWACWGGWTGAACWGTWTAYCCYCC

mlCOIint_Tag3

ACTAGCGGWACWGGWTGAACWGTWTAYCCYCC

mlCOIint_Tag4

ACAGTCGGWACWGGWTGAACWGTWTAYCCYCC

mlCOIint_Tag5

ATCGACGGWACWGGWTGAACWGTWTAYCCYCC

mlCOIint_Tag6

ATGTCGGGWACWGGWTGAACWGTWTAYCCYCC

mlCOIint_Tag7

ATAGCAGGWACWGGWTGAACWGTWTAYCCYCC

jgHCO_Tag1

AGACGCTAIACYTCIGGRTGICCRAARAAYCA

jgHCO_Tag2

AGTGTATAIACYTCIGGRTGICCRAARAAYCA

jgHCO_Tag3

ACTAGCTAIACYTCIGGRTGICCRAARAAYCA

jgHCO_Tag4

ACAGTCTAIACYTCIGGRTGICCRAARAAYCA

jgHCO_Tag5

ATCGACTAIACYTCIGGRTGICCRAARAAYCA

jgHCO_Tag6

ATGTCGTAIACYTCIGGRTGICCRAARAAYCA

jgHCO_Tag7

ATAGCATAIACYTCIGGRTGICCRAARAAYCA

 

2- Amplicon library preparation

 

3- Illumina TruSeq DNA Sample Preparation

Procedures:

1- Amplicon library preparation

For better PCR success, we recommend diluting the gDNA to 10-ng/μl. Each sample will be amplified in triplicates (3 x 20 μl reactions per sample). We will prepare a negative control for each tailed primer set used.

A hierarchical tagging approach will be used with a combination of tailed PCR primers and Illumina Indexes. For example: if you are analyzing 9 ARMS and 3 fractions per ARMS, you will have 27 samples. You can use 9 Illumina Indexes and 3 tagged primer pairs per index. In that case you will prepare three PCR master mixes for primer set Tag1, Tag2 and Tag3.

 

  1. Prepare the appropriate number of PCR master mixes depending on the number of tagged primer sets used. If using three as in the above example, prepare three master mixes, one for each tagged primer set. Calculate the number of samples per primer set and multiply by three (each sample will be run in triplicates). Add enough reagents to also run a negative control for each tagged primer set. For the negative control, replace the DNA with PCR grade water.
  2. Dilute each primer set to 10 μM.
  3. Prepare PCR master Mixes:

Reagent

Volume (μL)

PCR grade water

12.2

10X Advantage 2 PCR Buffer

2

mlCOIint_Tagx (10 μM)

1

jgHCO_Tagx (10 μM)

1

50X dNTP Mix (10 mM ea.)

1.4

50X Advantage 2 Polymerase Mix

0.4

Sample gDNA or water for neg. control

2

 

 

Final volume

20

 

  1. Run thermocycler with the following, touch-down, PCR profile:

Temperature (oC)

Time

Cycles #

95

1 mn

 

95

10 s

 

62

30 s

16

72

60 s

 

95

10 s

 

46

30 s

20

72

60 s

 

72

7 mn

 

4

 

 

  1. Run each PCR on an agarose gel to check PCR success. The band observed should be ~365-380 bp.
  2. Combine successful triplicate PCR reactions for each sample into a single tube (do not yet pool samples together at this point).
  3. Quantify amplicons using Qubit® dsDNA HS Assay Kit or Quant-iT PicoGreen dsDNA Assay Kit (ThermoFisher/Invitrogen) following manufacturer’s instructions.
  4. For each Illumina Index you will use downstream, pool 1μg of each tagged PCR sample into a single clean tube (or an equal amount to totalize >1μg for each index). Be careful that each single Illumina Index (ligated below) contains only samples tagged with different PCR primers. For example, for Index AD002 we will pool libraries tagged with PCR primers tag 1, 2 and 3. We can use those same tags for each Index used but within an index those tags must be different in order to bioinformatically separate the different libraries.
  5. Clean the amplicon’s pool using AMPure XP beads, following manufacturer’s instructions.
  6. Quantify each amplicon’s pool with Qubit® dsDNA HS Assay Kit or Quant-iT PicoGreen dsDNA Assay Kit.
  7. Transfer 1μg in a 0.5ml tube or PCR plate.

// Safe stopping point // You can freeze the pool of amplicons at -20oC //

 

2- Illumina TruSeq DNA Sample Preparation

For this step, we are following the Illumina TruSeq DNA LT Sample Preparation protocol. If you are not using all 12 Illumina Indexes in a single run, make sure to choose the indices according to the Low Plex Pooling guidelines described on page 45 of the protocol.

Because we are starting with pooled PCR products, we will start the protocol at the Perform End Repair step (page 63).

Follow carefully the instructions provided in the Illumina TruSeq DNA LT Sample Preparation protocol as the protocol below only contains headlines of the process.

*Note: If, for the bead cleanup, you will use a magnetic rack and not a magnetic plate, you will have to transfer the samples to 1.5-mL tubes before each cleanup.

 

  1. Perform End Repair (Page 63): Bring the volume of each pooled amplicon to 60μL with provided Resuspension Buffer.
  2. Add 40 μl of End Repair Mix 2 to each tube and mix thoroughly.
  3. Incubate the samples in the thermocycler at 30°C for 30 minutes.
  4. Proceed to Cleanup using the AMPure XP beads as described on Page 64. The final volume after cleanup step is 15 μl.

// safe stopping point // You can freeze the sample at -20oC //

  1. Adenylate 3’ Ends (Page 67):  Add 2.5 μl of Resuspension Buffer to each pooled amplicon.
  2. Add 12.5ml of A-Tailing Mix to each sample and mix thoroughly.
  3. Incubate the samples in the thermocycler at 37°C for 30 minutes.
  4. Ligate Adapters (page 69):

        Choose the adapters using the guidelines provided page 45 of the Illumina Illumina TruSeq DNA LT Sample Preparation protocol.

  1. Add 2.5ml of Resuspension Buffer to each sample.
  2. Add 2.5μl of Ligation Mix (directly from the freezer and put back immediately).
  3. Carefully add 2.5μl of corresponding adapter to each sample and mix thoroughly.
  4. Incubate the samples in the thermocycler at 30°C for 30 minutes.
  5. Remove the samples and add 5ml of Stop Ligation Buffer. Mix thoroughly.
  6. Proceed to Cleanup using the AMPure XP beads as described on Page 73-75. The final volume after cleanup step is 20 μl.

// safe stopping point // You can freeze the sample at -20oC //

  1. Quantify the libraries using Qubit® dsDNA HS Assay Kit or Quant-iT PicoGreen dsDNA Assay Kit following manufacturer’s instructions.
  2. Pool 50ng of each library into a single tube and mix thoroughly.
  3. Quantify the mixed library pool as in step 15.
  4. Validate the library with qPCR. We use KAPA Library Quantification Kit for Illumina platforms.
  5. Prepare DNA template for sequencing. Use the MiSeq Reagent kit v3 (600 cycles).

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