Evaluation and recommendation of sample Evaluation and recommendation of sample preservation techniques for 16S rRNA sequencing in preservation techniques for 16S rRNA sequencing in oilfield systemspreservation techniques for 16S rRNA sequencing in oilfield systemsoilfield systems

Jordan J. Schmidt1, Brad McIlwain1, Wei Huang1, Gilberto Guerrero2, Prasad Dhulipala2, Mary Eid2, Neil Sharma1Jordan J. Schmidt1, Brad McIlwain1, Wei Huang1, Gilberto Guerrero2, Prasad Dhulipala2, Mary Eid2, Neil Sharma1

1LuminUltra Technologies Ltd, 520 King Street, Fredericton, NB, Canada, E3B 6G32Baker Hughes, Houston, TX2Baker Hughes, Houston, TX

BACKGROUNDBACKGROUND

The implementation of metagenomics in oil and gas industry has drastically increased in the last several years. It is being used for a

BACKGROUNDBACKGROUND

The implementation of metagenomics in oil and gas industry has drastically increased in the last several years. It is being used for a host of applications, including: detection of the origin of microbially influenced corrosion (MIC), biocide efficacy studies, and host of applications, including: detection of the origin of microbially influenced corrosion (MIC), biocide efficacy studies, and microbial content evaluations of injection water and water used for hydraulic fracturing. One major deficiency with the application of metagenomics, is the lack of standardized protocols. Due to the remote nature of oilfields, sample preservation and maintaining metagenomics, is the lack of standardized protocols. Due to the remote nature of oilfields, sample preservation and maintaining integrity of microbial population during transport is of particular concern. If not preserved properly, the microbial community composition can change between sample collection and DNA extraction.integrity of microbial population during transport is of particular concern. If not preserved properly, the microbial community composition can change between sample collection and DNA extraction.

OBJECTIVEOBJECTIVEOBJECTIVEOBJECTIVE

To evaluate several common preservation techniques that are suitable for remote oilfield locations and contribute towards the To evaluate several common preservation techniques that are suitable for remote oilfield locations and contribute towards the development of standard protocols for metagenomics analysis.development of standard protocols for metagenomics analysis.

METHODOLOGYMETHODOLOGYMETHODOLOGYMETHODOLOGY

Two samples were collected from an oilfield and shipped to the lab. Once received by the lab, the DNA was extracted from the control sample. Samples were then preserved using various methods and Two samples were collected from an oilfield and shipped to the lab. Once received by the lab, the DNA was extracted from the control sample. Samples were then preserved using various methods and subjected to different scenarios including a simulated shipping delay and temperature cycling. Preserved samples were held for 7 and 14 days prior to DNA extraction and analysis. All samples were analyzed for total prokaryotes using qPCR and sequenced using Illumina 16s rRNA sequencing (515/806 primer).for total prokaryotes using qPCR and sequenced using Illumina 16s rRNA sequencing (515/806 primer).

1. Control2. LuminUltra GeneCount Chemical Preservation (7, 14 days)

qPCR – Total Prokaryotes

16S rRNASequencing

Well Water Frac Pit Water2. LuminUltra GeneCount Chemical Preservation (7, 14 days)3. Filtered and dried (7, 14 days)

Prokaryotes Sequencing

Temperature cycled (12 h at 20°C, 12h at 40°C)3. Filtered and dried (7, 14 days)4. Unpreserved (7, 14 days)5. Refrigerated (7, 14 days)

Temperature cycled (12 h at 20°C, 12h at 40°C)5. Refrigerated (7, 14 days)6. Frozen (14 days)6. Frozen (14 days)7. Shipping delay (Refrigerated for 24 hours, room temperature for 3 days)

Sample Collection Sample Preservation Sample AnalysisSample Collection Sample Preservation Sample Analysis

Two samples were also preserved in the field using LuminUltra’s GeneCount chemical preservation and by filtering and air drying. Preserved samples were shipped to the lab where they were further Two samples were also preserved in the field using LuminUltra’s GeneCount chemical preservation and by filtering and air drying. Preserved samples were shipped to the lab where they were further temperature cycled and analyzed after 7 and 14 days.

RESULTS – IN LABRESULTS – IN LAB RESULTS– IN FIELDRESULTS– IN FIELDRESULTS – IN LABRESULTS – IN LAB RESULTS– IN FIELDRESULTS– IN FIELD

Well Water Frac Pit Water Well WaterWell Water Frac Pit Water

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OUTCOMES – IN FIELDOUTCOMES – IN FIELD

1. Well Water – Similar to the laboratory results, filtration and GeneCount chemical preservation

OUTCOMES – IN FIELDOUTCOMES – IN FIELD

1. Well Water – Similar to the laboratory results, filtration and GeneCount chemical preservation gave similar results for both hold times. Compared to each other the preservation methods

High Similarity High Similarity

gave similar results for both hold times. Compared to each other the preservation methods were also quite similar.

2. Frac Pit Water – The GeneCount chemical preservation kit gave similar results after 7 and 14 High Similarity High Similarity 2. Frac Pit Water – The GeneCount chemical preservation kit gave similar results after 7 and 14 days of temperature cycling. Like the laboratory results, the filtered samples had low similarity days of temperature cycling. Like the laboratory results, the filtered samples had low similarity when compared to each other and compared to the chemically preserved samples. The filtered samples were characterized by relative increases in Pseudomonas (day 7), Bacillus (day 7 and OUTCOMES – IN LABOUTCOMES – IN LAB samples were characterized by relative increases in Pseudomonas (day 7), Bacillus (day 7 and 14) and Streptomyces (day 7 and 14) compared to the chemically preserved samples.

OUTCOMES – IN LABOUTCOMES – IN LAB

1. There were three preservation techniques that gave similar results to the control in both

14) and Streptomyces (day 7 and 14) compared to the chemically preserved samples.

CONCLUSIONSCONCLUSIONS1. There were three preservation techniques that gave similar results to the control in both

samples: freezing, refrigeration and GeneCount chemical preservation. 2. Filtering and drying had similar results to the control in well water (lower alpha diversity) but

CONCLUSIONSCONCLUSIONS2. Filtering and drying had similar results to the control in well water (lower alpha diversity) but

not in frac pit water (higher alpha diversity), which shows that it does not universally preserve Various preservation methods were tested on two oilfield sample matrices. Three preservation not in frac pit water (higher alpha diversity), which shows that it does not universally preserve all samples. There was a considerable increase in Bacilli-associated genera in the filtered frac pit water.

Various preservation methods were tested on two oilfield sample matrices. Three preservation methods performed well for both samples: freezing, refrigeration and GeneCount chemical preservation. Filtration and air drying worked well for one sample, but not the other. This shows pit water.

3. The GeneCount chemical preservation kit did result in some additional low abundance OTUs,

preservation. Filtration and air drying worked well for one sample, but not the other. This shows that testing multiple sample types is important when evaluating preservation methods.3. The GeneCount chemical preservation kit did result in some additional low abundance OTUs,

especially in the highly turbid well water. It is hypothesized that this is due to the extended extraction period offered by chemical preservation which resulted in the lysing of additional

that testing multiple sample types is important when evaluating preservation methods.

The results show that chemical sample preservation is recommended, The results show that chemical sample preservation is recommended, extraction period offered by chemical preservation which resulted in the lysing of additional and more robust cells.

The results show that chemical sample preservation is recommended, particularly in cases where cold-chain transport is not possible.

The results show that chemical sample preservation is recommended, particularly in cases where cold-chain transport is not possible.and more robust cells. particularly in cases where cold-chain transport is not possible.particularly in cases where cold-chain transport is not possible.