1st Experiment 24/11/16

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See 2016_11_24_Miseq_Sheet

1. Sanger Sequencing

Plate #4 - 25mg of Tissue was extracted by AGRF. DNA was diluted to 5ng/ul.

Samples were sanger sequenced with 16SAR (Palumbi) primer. If they failed, I used COI3 cocktail (Ivanova). FASTA sequences from Plate 4 are in the folder named Sanger Sequence FASTA Plate #4.

Naming - Plate position, primer, sample ID. ie reater than A1-16S-AR_1952.

2. DNA and Tissue Pools of Plate 4

We wanted to explore the possibility of using a metabarcoding approach.

For metabarcoding we re-examined specimens already identified from sanger sequences. We mixed DNA from many samples (n=16 or n=96) and did a single amplification (i.e. up to 96 DNA extractions processed in a

single-tube marker amplification). We also took it a step further and tried blending a set

amount of tissue from many fish specimens (n=16 or n=96) and did a single DNA extraction

on the tissue mixes (i.e. a single DNA extraction and single tube amplification for up to 96

samples). See 2016_11_24_Miseq_Sheet for DNA and Tissue Pool mixes.

3. Miseq Run

16 samples were ran on a 250bp pe read. Each sample was amplified with 3 primer sets - COI (please note one dual labelled set was used), 12s and 16s (Primers listed on 2016_11_24_Miseq_Sheet). They were diluted 1:10 and illumina sequencing adaptors were added (please note I used same I7 and I5 per sample, so they had to be sorted on amplicon).

2016_11_24_fastq_files has the data from miseq. and 2016_11_24_merged_fastq_files has the merged files.

For some unknown reason 16s tissue produced no data.

2nd Experiment 04/07/17

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1. DNA Extractions

Plate #1, 2 and 3 - 25mg of Tisse was extracted by AGRF. DNA was diluted to 5ng/ul. We also used Plate #4 from experiment above. See Plate Layout for sample allocation.

2. Tissue and DNA Pools

DNA pools were from Plate 1, 2, 3 and 4.

Tissue Mixes were from Plate 2 and 4 only.

We wanted to explore the possibility of using a metabarcoding approach.

We mixed DNA from many samples (n=16 or n=96) and did a single amplification (i.e. up to 96 DNA extractions processed in a single-tube marker amplification). We also took it a step further and tried blending a set

amount of tissue from many fish specimens (n=16 or n=96) and did a single DNA extraction

on the tissue mixes (i.e. a single DNA extraction and single tube amplification for up to 96

samples). See plate layout for DNA and Tissue Pool mixes.

3. Miseq Run

577 samples were sequenced in a 250bp pe read. See 2017_07_04_Miseq Sheet.

Plate 1, 2 3 and 4 were all sequenced with Leray Primers.(Please note I accidentally amplified the first half of plate one with one pair of dual labelled COI primers, index on miseq sheet).

I also made a plate of tissue and DNA pools (see plate layout for DNA and Tissue Pool mixes) and amplified those with 4 primers (primer sequences on miseq sheet)

COI (individual dual labelled primers, 1st round index are on miseq sheet)

12s Fish

16s Chordate

NADH

The last 4 samples with 12s were to add to database as there are no 12S sequences for those species on genbank.

See PCR recipes for annealing temp and cycling etc

I accidentally put the marker under sample name so the original sample ID was lost and miseq gave it a new name (name from miseq output) and then another new name from merged file. Finally I gave them a unique sample ID. See name file if you need more information.

2017_07_04 has the data from miseq. and 2017_07_04_merged_fastq_files has the merged files.

Samples were clustered using zero radius OTU's.

4.Results

See Results database.

The spreadsheet has all of the possible name combinations from the run. It also contains the Haul ID and date, time, lat, long etc. There is a morph taxa ID which refers to what the observer has identified the fish and then there is Seq_Taxa_ID which is the sequencing result. There is also a list of primers that were used to identify the fish. 0 indicated that the primer wasnt used, 1 indicates it was. The second tab has all of the info for the samples that failed.

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