16S long-read nanopore sequencing of human endometrial microbiome
Samples for this study were obtained from leftover endometrial tissue from routinely performed endometrial scratching of women after repeated implantation failure. The study was approved by the ethics committee of the Medical University of Vienna (ID: EK-1181/2019). For sampling, an endometrial suction curette (Probet, Gynetics, Belgium) was inserted into the uterine cavity without touching the vaginal wall. Tissue was placed into a sterile tubed and immediately stored at -20°C for maximum of two weeks. DNA was extracted using PureLink™ Microbiome DNA Purification Kit (Thermo Fisher Scientific, Carlsbad, CA, USA) according to the protocol. As positive control, Zymo Microbiome Community Standard was utilized. Here, either bacterial DNA was extracted using PureLink™ Microbiome DNA Purification Kit (Thermo Fisher Scientific, 'Zymo ex. T') or DNeasy Blood & Tissue kit (QIAGEN, Hilden, Germany, 'Zymo ex. Q'), or the Zymo DNA Community Standard ('Zymo DNA') was utilized for PCR amplification. As negative control, deionized water was included into the entire workflow to determine contaminations from exterior sources or from the utilized kits.16S rRNA region from different DNA samples was amplified using the 16S Barcoding Kit SQK-RAB204 from Oxford Nanopore Technologies (ONT) (27F and 1492R primer sequences). Amplified, purified and pre-processed DNA was sequenced using MinION sequencer from ONT. Live basecalling was performed using Guppy provided by the MinKNOW platform (ONT). Demultiplexing and bacterial taxonomy assignment was performed with the 16S workflow provided by the EPI2ME platform (ONT; https://epi2me.nanoporetech.com), based on the NCBI 16S bacterial database. The here uploaded fastq files are derived from the EPI2ME workflow after passing minimum quality requirements (Q-score > 9). Barcode sequences are included in the fastq sequences and can be demultiplexed as followed (according tohttps://community.nanoporetech.com/technical_documents/chemistry-technical-document/v/chtd_500_v1_revm_07jul2016/barcoding-kits):BC 01 AAGAAAGTTGTCGGTGTCTTTGTGBC 02 TCGATTCCGTTTGTAGTCGTCTGTBC 03 GAGTCTTGTGTCCCAGTTACCAGGBC 04 TTCGGATTCTATCGTGTTTCCCTABC 05 CTTGTCCAGGGTTTGTGTAACCTTBC 06 TTCTCGCAAAGGCAGAAAGTAGTCBC 07 GTGTTACCGTGGGAATGAATCCTTBC 08 TTCAGGGAACAAACCAAGTTACGTBC 09 AACTAGGCACAGCGAGTCTTGGTTBC 10 AAGCGTTGAAACCTTTGTCCTCTCBC 11 GTTTCATCTATCGGAGGGAATGGABC 12 CAGGTAGAAAGAAGCAGAATCGGAThe following samples with the respective barcode from the publication '16S long-read nanopore sequencing is feasible and reliable for endometrial microbiome analysis' can be found in these files: Sequencing run ID date Sample ID/control ID Barcode (BC)186724 12.12.18 H2O 1 01186724 12.12.18 Zymo DNA 1 03186724 12.12.18 Zymo ex. T 1 02186724 12.12.18 3 06186724 12.12.18 5 08188961 10.01.19 H2O 2 01188961 10.01.19 Zymo DNA 2 04188961 10.01.19 Zymo ex. Q 1 05188961 10.01.19 6 06188961 10.01.19 7 07188961 10.01.19 8 08188961 10.01.19 9 09192897 28.02.19 Zymo DNA 3 01192897 28.02.19 Zymo spike-in 1 (Neisseria) 02192897 28.02.19 Zymo spike-in 2 (Streptococcus) 03192897 28.02.19 Zymo ex. Q 2 05192897 28.02.19 Zyme ex. T 2 04192897 28.02.19 1 08192897 28.02.19 2 09193429 07.03.19 13 04193429 07.03.19 16 06193429 07.03.19 17 09193429 07.03.19 20 05193429 07.03.19 21 12193429 07.03.19 22 07193429 07.03.19 23 08199345 30.04.19 H2O 3 01199345 30.04.19 Mycoplasma swab 1 11199345 30.04.19 Mycoplasma swab 2 12199345 30.04.19 14 02199345 30.04.19 15 03199345 30.04.19 18 04199345 30.04.19 26 07199345 30.04.19 27 08199345 30.04.19 28 09199345 30.04.19 29 10200679 14.05.19 H2O 4 01200679 14.05.19 12 05200679 14.05.19 31 07200679 14.05.19 33 11The here uploaded files are named according to the sequencing dates, e.g. 20181212.fastq.gz for all reads from 12.12.18 and 20190110.fastq,gz for those from 10.01.19.