Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples

Abstract The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S ribosomal RNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibili...
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Autor*in:	Julia A. Schwenker [verfasserIn] Meike Friedrichsen [verfasserIn] Silvio Waschina [verfasserIn] Corinna Bang [verfasserIn] Andre Franke [verfasserIn] Ricarda Mayer [verfasserIn] Christina S. Hölzel [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2022

Schlagwörter:	bovine milk DNA extraction Illumina amplicon sequencing milk microbiome

Übergeordnetes Werk:	In: MicrobiologyOpen - Wiley, 2012, 11(2022), 2, Seite n/a-n/a
Übergeordnetes Werk:	volume:11 ; year:2022 ; number:2 ; pages:n/a-n/a

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.1002/mbo3.1275

Katalog-ID:	DOAJ086521926

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520			\|a Abstract The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S ribosomal RNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony‐forming units [cfu] and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. Different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were resequenced in triplicates (n = 120). The most reproducible results for α‐ and β‐diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pretreatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pretreatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols.
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700	0		\|a Silvio Waschina \|e verfasserin \|4 aut
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700	0		\|a Andre Franke \|e verfasserin \|4 aut
700	0		\|a Ricarda Mayer \|e verfasserin \|4 aut
700	0		\|a Christina S. Hölzel \|e verfasserin \|4 aut
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912			\|a GBV_ILN_4046
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912			\|a GBV_ILN_4325
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allfields	10.1002/mbo3.1275 doi (DE-627)DOAJ086521926 (DE-599)DOAJc82dac7ca02747239355bc30be96c633 DE-627 ger DE-627 rakwb eng QR1-502 Julia A. Schwenker verfasserin aut Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S ribosomal RNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony‐forming units [cfu] and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. Different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were resequenced in triplicates (n = 120). The most reproducible results for α‐ and β‐diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pretreatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pretreatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols. bovine milk DNA extraction Illumina amplicon sequencing milk microbiome Microbiology Meike Friedrichsen verfasserin aut Silvio Waschina verfasserin aut Corinna Bang verfasserin aut Andre Franke verfasserin aut Ricarda Mayer verfasserin aut Christina S. Hölzel verfasserin aut In MicrobiologyOpen Wiley, 2012 11(2022), 2, Seite n/a-n/a (DE-627)718621158 (DE-600)2661368-2 20458827 nnns volume:11 year:2022 number:2 pages:n/a-n/a https://doi.org/10.1002/mbo3.1275 kostenfrei https://doaj.org/article/c82dac7ca02747239355bc30be96c633 kostenfrei https://doi.org/10.1002/mbo3.1275 kostenfrei https://doaj.org/toc/2045-8827 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_636 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2106 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2118 GBV_ILN_2122 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2470 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 11 2022 2 n/a-n/a
spelling	10.1002/mbo3.1275 doi (DE-627)DOAJ086521926 (DE-599)DOAJc82dac7ca02747239355bc30be96c633 DE-627 ger DE-627 rakwb eng QR1-502 Julia A. Schwenker verfasserin aut Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S ribosomal RNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony‐forming units [cfu] and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. Different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were resequenced in triplicates (n = 120). The most reproducible results for α‐ and β‐diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pretreatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pretreatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols. bovine milk DNA extraction Illumina amplicon sequencing milk microbiome Microbiology Meike Friedrichsen verfasserin aut Silvio Waschina verfasserin aut Corinna Bang verfasserin aut Andre Franke verfasserin aut Ricarda Mayer verfasserin aut Christina S. Hölzel verfasserin aut In MicrobiologyOpen Wiley, 2012 11(2022), 2, Seite n/a-n/a (DE-627)718621158 (DE-600)2661368-2 20458827 nnns volume:11 year:2022 number:2 pages:n/a-n/a https://doi.org/10.1002/mbo3.1275 kostenfrei https://doaj.org/article/c82dac7ca02747239355bc30be96c633 kostenfrei https://doi.org/10.1002/mbo3.1275 kostenfrei https://doaj.org/toc/2045-8827 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_636 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2106 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2118 GBV_ILN_2122 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2470 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 11 2022 2 n/a-n/a
allfields_unstemmed	10.1002/mbo3.1275 doi (DE-627)DOAJ086521926 (DE-599)DOAJc82dac7ca02747239355bc30be96c633 DE-627 ger DE-627 rakwb eng QR1-502 Julia A. Schwenker verfasserin aut Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S ribosomal RNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony‐forming units [cfu] and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. Different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were resequenced in triplicates (n = 120). The most reproducible results for α‐ and β‐diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pretreatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pretreatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols. bovine milk DNA extraction Illumina amplicon sequencing milk microbiome Microbiology Meike Friedrichsen verfasserin aut Silvio Waschina verfasserin aut Corinna Bang verfasserin aut Andre Franke verfasserin aut Ricarda Mayer verfasserin aut Christina S. Hölzel verfasserin aut In MicrobiologyOpen Wiley, 2012 11(2022), 2, Seite n/a-n/a (DE-627)718621158 (DE-600)2661368-2 20458827 nnns volume:11 year:2022 number:2 pages:n/a-n/a https://doi.org/10.1002/mbo3.1275 kostenfrei https://doaj.org/article/c82dac7ca02747239355bc30be96c633 kostenfrei https://doi.org/10.1002/mbo3.1275 kostenfrei https://doaj.org/toc/2045-8827 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_636 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2106 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2118 GBV_ILN_2122 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2470 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 11 2022 2 n/a-n/a
allfieldsGer	10.1002/mbo3.1275 doi (DE-627)DOAJ086521926 (DE-599)DOAJc82dac7ca02747239355bc30be96c633 DE-627 ger DE-627 rakwb eng QR1-502 Julia A. Schwenker verfasserin aut Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S ribosomal RNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony‐forming units [cfu] and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. Different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were resequenced in triplicates (n = 120). The most reproducible results for α‐ and β‐diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pretreatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pretreatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols. bovine milk DNA extraction Illumina amplicon sequencing milk microbiome Microbiology Meike Friedrichsen verfasserin aut Silvio Waschina verfasserin aut Corinna Bang verfasserin aut Andre Franke verfasserin aut Ricarda Mayer verfasserin aut Christina S. Hölzel verfasserin aut In MicrobiologyOpen Wiley, 2012 11(2022), 2, Seite n/a-n/a (DE-627)718621158 (DE-600)2661368-2 20458827 nnns volume:11 year:2022 number:2 pages:n/a-n/a https://doi.org/10.1002/mbo3.1275 kostenfrei https://doaj.org/article/c82dac7ca02747239355bc30be96c633 kostenfrei https://doi.org/10.1002/mbo3.1275 kostenfrei https://doaj.org/toc/2045-8827 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_636 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2106 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2118 GBV_ILN_2122 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2470 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 11 2022 2 n/a-n/a
allfieldsSound	10.1002/mbo3.1275 doi (DE-627)DOAJ086521926 (DE-599)DOAJc82dac7ca02747239355bc30be96c633 DE-627 ger DE-627 rakwb eng QR1-502 Julia A. Schwenker verfasserin aut Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S ribosomal RNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony‐forming units [cfu] and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. Different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were resequenced in triplicates (n = 120). The most reproducible results for α‐ and β‐diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pretreatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pretreatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols. bovine milk DNA extraction Illumina amplicon sequencing milk microbiome Microbiology Meike Friedrichsen verfasserin aut Silvio Waschina verfasserin aut Corinna Bang verfasserin aut Andre Franke verfasserin aut Ricarda Mayer verfasserin aut Christina S. Hölzel verfasserin aut In MicrobiologyOpen Wiley, 2012 11(2022), 2, Seite n/a-n/a (DE-627)718621158 (DE-600)2661368-2 20458827 nnns volume:11 year:2022 number:2 pages:n/a-n/a https://doi.org/10.1002/mbo3.1275 kostenfrei https://doaj.org/article/c82dac7ca02747239355bc30be96c633 kostenfrei https://doi.org/10.1002/mbo3.1275 kostenfrei https://doaj.org/toc/2045-8827 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_636 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2106 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2118 GBV_ILN_2122 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2470 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 11 2022 2 n/a-n/a
language	English
source	In MicrobiologyOpen 11(2022), 2, Seite n/a-n/a volume:11 year:2022 number:2 pages:n/a-n/a
sourceStr	In MicrobiologyOpen 11(2022), 2, Seite n/a-n/a volume:11 year:2022 number:2 pages:n/a-n/a
format_phy_str_mv	Article
institution	findex.gbv.de
topic_facet	bovine milk DNA extraction Illumina amplicon sequencing milk microbiome Microbiology
isfreeaccess_bool	true
container_title	MicrobiologyOpen
authorswithroles_txt_mv	Julia A. Schwenker @@aut@@ Meike Friedrichsen @@aut@@ Silvio Waschina @@aut@@ Corinna Bang @@aut@@ Andre Franke @@aut@@ Ricarda Mayer @@aut@@ Christina S. Hölzel @@aut@@
publishDateDaySort_date	2022-01-01T00:00:00Z
hierarchy_top_id	718621158
id	DOAJ086521926
language_de	englisch
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callnumber-first	Q - Science
author	Julia A. Schwenker
spellingShingle	Julia A. Schwenker misc QR1-502 misc bovine milk misc DNA extraction misc Illumina amplicon sequencing misc milk microbiome misc Microbiology Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples
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topic_title	QR1-502 Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples bovine milk DNA extraction Illumina amplicon sequencing milk microbiome
topic	misc QR1-502 misc bovine milk misc DNA extraction misc Illumina amplicon sequencing misc milk microbiome misc Microbiology
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title	Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples
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title_full	Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples
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author_browse	Julia A. Schwenker Meike Friedrichsen Silvio Waschina Corinna Bang Andre Franke Ricarda Mayer Christina S. Hölzel
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title_sort	bovine milk microbiota: evaluation of different dna extraction protocols for challenging samples
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title_auth	Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples
abstract	Abstract The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S ribosomal RNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony‐forming units [cfu] and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. Different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were resequenced in triplicates (n = 120). The most reproducible results for α‐ and β‐diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pretreatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pretreatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols.
abstractGer	Abstract The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S ribosomal RNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony‐forming units [cfu] and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. Different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were resequenced in triplicates (n = 120). The most reproducible results for α‐ and β‐diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pretreatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pretreatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols.
abstract_unstemmed	Abstract The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S ribosomal RNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony‐forming units [cfu] and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. Different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were resequenced in triplicates (n = 120). The most reproducible results for α‐ and β‐diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pretreatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pretreatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols.
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title_short	Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples
url	https://doi.org/10.1002/mbo3.1275 https://doaj.org/article/c82dac7ca02747239355bc30be96c633 https://doaj.org/toc/2045-8827
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author2	Meike Friedrichsen Silvio Waschina Corinna Bang Andre Franke Ricarda Mayer Christina S. Hölzel
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Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony‐forming units [cfu] and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. Different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were resequenced in triplicates (n = 120). The most reproducible results for α‐ and β‐diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pretreatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pretreatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. 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Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples

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