human reproductive system metagenome
Zymo kit microbial DNA extraction from ThinPrep of subject 02
collection_date
2017-04-10
env_broad_scale
uterus(UBERON:0000995)
env_local_scale
uterine cervix(UBERON:0000002)
env_medium
Liquid Based Cytology(NCIT:C99177)
geo_loc_name
USA:Arkansas,Little Rock (GAZ,00165676)
host
Homo sapiens
lat_lon
not applicable
ethnicity
Caucasian
host_age
33.4
host_body_product
cervical mucus(UBERON:0000316)
host_sex
female
host_subject_id
Subject_02
samp_collect_device
ThinPrep Holologic, Marlborough, MA)
samp_mat_process
Liquid-based cytology (LBC) specimens were obtained from 20 patients with enrolled in a Phase II clinical trial of an HPV therapeutic vaccine (NCT02481414). In order to be eligible, participants had to have high grade squamous intraepithelial lesions (HSILs) or cannot rule out HSILs in cervical cytology or CIN2/3 in cervical biopsy. Those who qualified for the study based on their cervical cytology underwent cervical biopsy, and they qualified for vaccination if the results were CIN2/3. The cervical cytology specimens in this current study were collected before the vaccination and reserved in the vial of the ThinPrep Pap Test (HOLOGIC) as per Ravilla et al. 2019 (doi:10.1177/1534735419893063). The storage period from sample collection to DNA extraction was 716 ± 105 days. Each LBC sample was dispensed into four separate 2 mL sterile collection tubes (dispensed sample volume = 500 μL). DNA was extracted from each specimen using one of four commercially available kits: ZymoBIOMICS DNA Miniprep Kit (Zymo Research, D4300), QIAamp PowerFecal Pro DNA Kit (QIAGEN, 51804), QIAamp DNA Mini Kit (QIAGEN, 51304), and IndiSpin Pathogen Kit (Indical Bioscience, SPS4104). Applied sample volume of ThinPrep solution was 300 μL for Zymo , 300 μL for PowerFecal Pro, and 200 μL for QIAamp Mini, and 300 μL for IndiSpin. DNA yield for each method was evaluated by spectrophotometer (Nanodrop One, Thermo Scientific). Positive control was mock vaginal microbial communities composed of a mixture of genomic DNA from the American Type Culture Collection (ATCC MSA1007). Negative control was the ThinPrep preservation solution as blank extraction (doi: 10.1186/s40168-017-0267-5). Controls and the extracted DNA were sent to Argonne National Laboratory (IL, USA) for amplification and sequencing of the 16S rRNA gene on an Illumina MiSeq sequencing platform. Paired-end reads from libraries with 250-bp inserts were generated for the V4 region using the barcoded primer set: 515FB: 5'-GTGYCAGCMGCCGCGGTAA-3' and 806RB: 5'-GGACTACNVGGGTWTCTAAT-3' (dois: 10.1038/nature24621, 10.3354/ame01753, 10.1111/1462-2920.13023, /10.1128/mSystems.00009-15, (http://www.earthmicrobiome.org/protocols-and-standards/16s/). MiSeq Reagent Kit v2 (2 × 150 cycles, MS-102-2002) was used.
samp_store_dur
716 ± 105 days
samp_store_temp
-80 C
source_material_id
cervical mucus(UBERON:0000316)
original_sample_labels
S022
BarcodeSequence
ATCAGCCAGCTC
ForwardPrimer
GTGTGYCAGCMGCCGCGGTAA
ReversePrimer
GGACTACNVGGGTWTCTAAT
DNA_extraction_kit
ZymoBIOMICS DNA Miniprep Kit (Zymo Research, D4300)
cervical_intraepithelial_neoplasia_grade
CIN2
hpv_genotypes
16,58,61,62,70,81
hpv16
Positive
hpv18
Negative
high_risk_hpv
Positive
BioSampleModel
MIMARKS.survey
BioSampleModel
MIGS/MIMS/MIMARKS.human-vaginal