Combined bacterial and fungal targeted amplicon sequencing of respiratory samples: Does the DNA extraction method matter?

High-throughput sequencing techniques were used to analyze the diversity of respiratory microbiota in health and disease. Despite the extensive data available on the respiratory tract bacterial microbiota, the remnants of the less studied fungal components. This is partly due to the technical problems associated with fungal metagenomics analysis. In this study, we compared two protocols of DNA extraction and amplification of two targets for the combined fungal bacteria and fungi that are targeted amplicon sequencing analysis of sputum microbiota.Six breathing, selected randomly from a sample routine Mondor Hospital (Créteil, France) and treated anonymously, were tested after a routine culture of bacteria and fungi.

Two of spiked Fumigati Aspergillus and Aspergillus Nigri (105 conidia / ml). After the mechanical lysis, DNA was extracted using the automatic extraction of QIAsymphony® (AQE) or users PowerSoil® MoBio extraction (MPE). DNA yield and purity compared. DNA extracted from sputum spiked into the target (i) real-time PCR for detection of Aspergillus DNA and (ii) a combination of primary targeting barcode metagenomic analysis for ITS1 and ITS2 fungi, and bacteria V1-V2 and V3-V4 region of the 16S. amplicon libraries were prepared using reagents MiSeq V3 kit on the Illumina platform. Data were analyzed using software PyroMIC © and Shaman, and compared with culture results.AQE extraction provides a higher yield of the (ratio AQE / MPE DNA = 4.5 [1.3 to 11]) DNA in a shorter period of time.

Results of Aspergillus DNA is detected by qPCR were similar to sputum spiked regardless of the extraction protocol. extraction quite an impact on biodiversity or the relative abundance of bacterial communities using targeted amplicon sequencing (2/43 taxa impact). For fungi, the relative abundance of major taxa 4/11 affected and AQE results were closer to the culture results. V1-V2 or V3-V4 and ITS1 or ITS2 target is judged diversity major taxa of bacteria and fungi, but ITS2 and V3-V4 detected smaller taxa.Our results show the importance of joint extraction of DNA for bacteria and fungi targeted metagenomics respiratory samples. Extraction protocol may affect the results of the DNA and the relative abundance of a few bacteria, but more fungal taxa. For the analysis of fungi, ITS2 allow detection of a large number of taxa small compared with ITS1.

ACTG1 human mutation associated with high frequency hearing loss, and patients with mutations in this gene are good candidates for the electric acoustic stimulation. To better understand the genetic etiology of hearing loss cases, massively parallel DNA sequencing performed on a 7048 Japanese probands related hearing loss. Among the 1,336 patients with autosomal dominant hearing loss, we identified 15 probands (1.1%) with 13 ACTG1 potentially pathogenic variant. Six new variant and seven were reported previously.

We collect and analyze detailed clinical features of these patients. The average level of development of pure-tone hearing worsening of the average for the four frequency is 1.7 dB / year of age 0-50 years, and all individuals over 60 years old has a severe hearing loss. To better understand the mechanisms underlying causes of disease, the intracellular localization of the wild-type and mutant gamma-actins checked using NIH / 3T3 fibroblast cell line

TMB Substrate, for Western Blot
F129-100 100 ml
EUR 249
Description: TMB Substrate, for Western Blot by Cygnus Technologies is available in Europe via Gentaur.

Hypersensitive Western Blot Chemiluminescent Substrate
CR0001-10 20mL (sufficient reagents for 4000 cm2 of membrane)
EUR 167

Hypersensitive Western Blot Chemiluminescent Substrate
CR0001-25 50mL (sufficient reagents for 10000 cm2 of membrane)
EUR 314

Hypersensitive Western Blot Chemiluminescent Substrate
CR0001-5 10mL (sufficient reagents for 2000 cm2 of membrane)
EUR 116

AgileBlotter? Automated Western Blot Processor
ACT-ABH-40
EUR 16939

Interferon alpha Western Blot Antibody
20-abx137014
  • EUR 356.00
  • EUR 523.00
  • 0.5 mg
  • 1 mg

Chemiluminescent Western Blot Kit (OKRA00001)
OKRA00001 1 kit
EUR 570
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

Chemiluminescent Western Blot Kit (OKRA00002)
OKRA00002 1 kit
EUR 570
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

Chemiluminescent Western Blot Kit (OKRA00003)
OKRA00003 1 kit
EUR 570
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

Chemiluminescent Western Blot Kit (OKRA00004)
OKRA00004 1 kit
EUR 570
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

Colorimetric Western Blot Kit (OKRA00014)
OKRA00014 1 kit
EUR 609
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

Colorimetric Western Blot Kit (OKRA00015)
OKRA00015 1 each
EUR 609
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

Colorimetric Western Blot Kit (OKRA00016)
OKRA00016 1 kit
EUR 609
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

Colorimetric Western Blot Kit (OKRA00017)
OKRA00017 1 kit
EUR 609
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

PDCD4 Western Blot kit (AWBK09045)
AWBK09045 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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ETS1 Western Blot kit (AWBK100604)
AWBK100604 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
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ERG Western Blot kit (AWBK100625)
AWBK100625 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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GABRP Western Blot kit (AWBK13034)
AWBK13034 10 reactions
EUR 647
Description:
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  • Species reactivity:
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HTR1A Western Blot kit (AWBK13041)
AWBK13041 10 reactions
EUR 647
Description:
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  • Species reactivity:
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RAB14 Western Blot kit (AWBK13107)
AWBK13107 10 reactions
EUR 647
Description:
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  • Species reactivity:
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TNRC4 Western Blot kit (AWBK31525)
AWBK31525 10 reactions
EUR 647
Description:
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  • Species reactivity:
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ESRRG Western Blot kit (AWBK31655)
AWBK31655 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
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GLI2 Western Blot kit (AWBK31885)
AWBK31885 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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PAX4 Western Blot kit (AWBK32064)
AWBK32064 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
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AHR Western Blot kit (AWBK32243)
AWBK32243 10 reactions
EUR 647
Description:
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  • Species reactivity:
  • Application:
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GLI1 Western Blot kit (AWBK32368)
AWBK32368 10 reactions
EUR 647
Description:
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  • Species reactivity:
  • Application:
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PAX7 Western Blot kit (AWBK32393)
AWBK32393 10 reactions
EUR 647
Description:
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  • Species reactivity:
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LHX6 Western Blot kit (AWBK32553)
AWBK32553 10 reactions
EUR 647
Description:
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  • Species reactivity:
  • Application:
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HIPK2 Western Blot kit (AWBK32586)
AWBK32586 10 reactions
EUR 647
Description:
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  • Species reactivity:
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PAX7 Western Blot kit (AWBK32742)
AWBK32742 10 reactions
EUR 647
Description:
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  • Species reactivity:
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ZFP67 Western Blot kit (AWBK32749)
AWBK32749 10 reactions
EUR 647
Description:
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  • Species reactivity:
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OLIG2 Western Blot kit (AWBK32753)
AWBK32753 10 reactions
EUR 647
Description:
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  • Species reactivity:
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ENG Western Blot kit (AWBK33068)
AWBK33068 10 reactions
EUR 647
Description:
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  • Species reactivity:
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SOX5 Western Blot kit (AWBK33323)
AWBK33323 10 reactions
EUR 647
Description:
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  • Species reactivity:
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SOX10 Western Blot kit (AWBK33326)
AWBK33326 10 reactions
EUR 647
Description:
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  • Species reactivity:
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RPS16 Western Blot kit (AWBK33535)
AWBK33535 10 reactions
EUR 647
Description:
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  • Species reactivity:
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ATP7A Western Blot kit (AWBK33797)
AWBK33797 10 reactions
EUR 647
Description:
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  • Species reactivity:
  • Application:
  • Assay info:
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A1BG Western Blot kit (AWBK33810)
AWBK33810 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity: Human
  • Application:
  • Assay info:
  • Sensitivity:

NPM1 Western Blot kit (AWBK34094)
AWBK34094 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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LIG4 Western Blot kit (AWBK34122)
AWBK34122 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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RNF2 Western Blot kit (AWBK34290)
AWBK34290 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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ZFP36 Western Blot kit (AWBK34385)
AWBK34385 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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ARIH2 Western Blot kit (AWBK34418)
AWBK34418 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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KEAP1 Western Blot kit (AWBK34728)
AWBK34728 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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CLIC1 Western Blot kit (AWBK35045)
AWBK35045 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
  • Sensitivity:

KCNN2 Western Blot kit (AWBK35094)
AWBK35094 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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CLCN3 Western Blot kit (AWBK35504)
AWBK35504 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
  • Sensitivity:

P2RX7 Western Blot kit (AWBK35518)
AWBK35518 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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TRPM3 Western Blot kit (AWBK35609)
AWBK35609 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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PITX2 Western Blot kit (AWBK35634)
AWBK35634 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
  • Sensitivity:

ILF2 Western Blot kit (AWBK35731)
AWBK35731 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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ONECUT2 Western Blot kit (AWBK35750)
AWBK35750 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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ZFP42 Western Blot kit (AWBK36244)
AWBK36244 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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ANXA1 Western Blot kit (AWBK36568)
AWBK36568 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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SPP1 Western Blot kit (AWBK36677)
AWBK36677 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
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  • Assay info:
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RUNX2 Western Blot kit (AWBK36678)
AWBK36678 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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ZBP1 Western Blot kit (AWBK36711)
AWBK36711 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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Bapx1 Western Blot kit (AWBK36826)
AWBK36826 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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G3BP Western Blot kit (AWBK37713)
AWBK37713 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
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TWIST1 Western Blot kit (AWBK37997)
AWBK37997 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
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ID4 Western Blot kit (AWBK38058)
AWBK38058 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
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SATB1 Western Blot kit (AWBK38208)
AWBK38208 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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EED Western Blot kit (AWBK38384)
AWBK38384 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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TARDBP Western Blot kit (AWBK38941)
AWBK38941 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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TARDBP Western Blot kit (AWBK38942)
AWBK38942 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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BRD7 Western Blot kit (AWBK39018)
AWBK39018 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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JMJD3 Western Blot kit (AWBK40102)
AWBK40102 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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SNRP70 Western Blot kit (AWBK40439)
AWBK40439 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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SSB Western Blot kit (AWBK40462)
AWBK40462 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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STAU1 Western Blot kit (AWBK40537)
AWBK40537 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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PCBP1 Western Blot kit (AWBK40630)
AWBK40630 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
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HNRPLL Western Blot kit (AWBK41102)
AWBK41102 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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MSI2 Western Blot kit (AWBK41112)
AWBK41112 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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HNRPA3 Western Blot kit (AWBK41195)
AWBK41195 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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WNT2B Western Blot kit (AWBK41254)
AWBK41254 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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GNB1L Western Blot kit (AWBK41328)
AWBK41328 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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SLC12A1 Western Blot kit (AWBK41388)
AWBK41388 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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TPM1 Western Blot kit (AWBK41392)
AWBK41392 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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CEACAM6 Western Blot kit (AWBK41504)
AWBK41504 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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SLC22A1 Western Blot kit (AWBK41516)
AWBK41516 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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STK11 Western Blot kit (AWBK41743)
AWBK41743 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
  • Sensitivity:

OTC Western Blot kit (AWBK41766)
AWBK41766 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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TSHR Western Blot kit (AWBK41870)
AWBK41870 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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RHOBTB1 Western Blot kit (AWBK41883)
AWBK41883 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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KRT13 Western Blot kit (AWBK42031)
AWBK42031 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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IDH3A Western Blot kit (AWBK42237)
AWBK42237 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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SLC1A5 Western Blot kit (AWBK42247)
AWBK42247 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
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PBEF1 Western Blot kit (AWBK42255)
AWBK42255 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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FST Western Blot kit (AWBK42287)
AWBK42287 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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SLC22A7 Western Blot kit (AWBK42306)
AWBK42306 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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ASPN Western Blot kit (AWBK42487)
AWBK42487 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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RSAD2 Western Blot kit (AWBK42696)
AWBK42696 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
  • Sensitivity:

PANX2 Western Blot kit (AWBK42778)
AWBK42778 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
  • Assay info:
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PANX1 Western Blot kit (AWBK42783)
AWBK42783 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
  • Application:
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TGFBI Western Blot kit (AWBK44268)
AWBK44268 10 reactions
EUR 647
Description:
  • Description of target:
  • Species reactivity:
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PTPN2 Western Blot kit (AWBK45362)
AWBK45362 10 reactions
EUR 647
Description:
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  • Species reactivity:
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