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A Quantitative Comparison Of Urine Centrifugation And Filtration For The Isolation And Analysis Of Urinary Nucleic Acid Biomarkers – Scientific Reports –

Study subjects and specimens

Urinary specimens were collected from kidney transplant recipients (KTxR) enrolled in the WCM IRB protocol 20-01021269 protocol entitled “Metagenomic profiling of urinary cell-free DNA to monitor urinary tract infection after kidney transplantation”, We studied 28 paired urine specimens from 16 KTxR, 12 KTxR provided specimens at 2 different time points and 4 provided at a single time point. The study cohort included 11 (69%) male and 10 (63%) living donor transplant recipients. The mean age of the study cohort was 50 ± 11.7 years. Urinary samples were collected at 46 ± 10.9 days post-transplant, and 46% (13/28) urine specimens were collected in the presence of a ureteral stent in the transplanted kidney. At the time of specimen collection, none of the patients had symptoms of urinary tract infection. All specimens and patient participation were approved under the Institutional Division of Human Subject Protection (IRB protocol 20-01021269). Informed consent was obtained from all participants. All experiments were performed in accordance with relevant guidelines and regulations.

Urine collection and processing

Patients were asked to provide mid-stream clean catch urine specimen. All urine specimens were either processed immediately or stored at 4 °C for up to 4 h after collection. After collecting 5 ml for urine culture using BD Vacutainer®, UA No Additive tubes and 5 ml for urinalysis using BD Vacutainer®, Urine Culture preservative tubes, we split the remaining urine into two samples of equal volume to compare the two methodologies for separating cell-free DNA and urinary cellular fractions. Our traditional centrifugation method6,12 used in the CTOT-04 trial was compared to our laboratory developed filtration method, Weill Cornell Hybrid Protocol (WCHP)13. To evaluate the performance of both methods in measuring microbial cfDNA, the urine supernatant collected after centrifugation at 2000g for 30 min using the in the centrifugation method was compared to the urine effluent collected after passing the thru ZRC GF™ filter using in the WCHP method. In parallel, the urinary cell mRNA obtained from cell pellet lysates in the centrifugation method were compared to the cell lysates obtained from the WCHP method. Preservative free cell-free DNA and cell lysates were stored at − 80 °C for down-stream analysis.

cfDNA isolation, sequencing, and quantification

We extracted cfDNA from the urine fluid using QIAGEN’s QIAamp Circulating Nucleic Acid Kit (QIAGEN Cat# 55114). We followed QIAGEN’s protocol for DNA extraction in 1 ml of urine. We used Invitrogen’s Qubit dsDNA HS Assay Kit (Invitrogen Cat# Q33231) and Invitrogen Qubit 2.0 according to the manufacturer’s protocol to quantify cfDNA abundance post-isolation and to calculate the total cfDNA abundance in the urine fluid. To find the measurement error, we performed multiple cfDNA isolations. We made technical replicates of the extracts for which we obtained the Qubit values. The error was calculated as the mean total cfDNA abundance plus or minus the standard deviation for all measurements.

cfDNA bisulfite conversion, single-stranded cfDNA isolation, and sequencing library preparation

cfDNA was extracted from urine using Norgen’s urine Cell-Free Circulating DNA Purification Midi Kit (Norgen Cat #56700). The protocol was adapted to allow bisulfite salt conversion of nucleic acids prior to extraction. Here, an aliquot of 520 µL of urine was centrifuged at 15,000 RPM for 5 min to pellet cellular and other solid debris. 500 µL of supernatant was transferred to a new 15 mL falcon tube containing 3.25 mL of ammonium bisulfite solution (Zymo Research, product #5030) and heated to 98 °C for 15 min. Samples were then kept at 54 °C for 90 min. Then, cfDNA extraction was performed using a commercially available column-based kit (Norgen Biotek, product #56700). Before cfDNA elution, 200 µL of L-Desulphonation buffer (Zymo Research, product #5030) was added to the columns for 20 min, followed by two washes with 200 µL absolute ethanol. DNA was then eluted to 32 μl according to manufacturer recommendations. A single-stranded library preparation was performed using the SRSLY Pico-Plus DNA NGS Library Preparation Base Kit (Claret Biosciences, Cat# CBS-K250B-24). Libraries were then sequenced on a Nextseq 550 Illumina sequencer with a 2 × 75 bp read length.

Alignment to the human genome

Adapter and low-quality bases from the reads were trimmed using BBDuk14 and aligned to the C-to-T and G-to-A converted human genome using Bismark15 (Bismark-0.22.1). PCR duplicates were removed using Bismark. We estimated bisulfite conversion efficiency by quantifying the rate of C[A/T/C] methylation in human-aligned reads (using Meth Pipe V3.4.3).

Metagenomic abundance estimation from sequencing data

The metagenomic analysis is performed as previously described1,3,4,16. Specific to SIFT-seq, read-level filtering of contaminants is performed by removing sequenced reads with 4 or more cytosines present or one methylated CpG dinucleotide (the latter represents unmapped, human-derived molecules). Species-level filtering based on the distribution of mapped reads is carried out by first aligning filtered and unfiltered datasets independently. Cytosine densities of mapping coordinates in both datasets are measured using custom scripts, and their distributions are compared using a Kolmogorov–Smirnov test. Significantly different filtered-unfiltered distributions are further processed (D-statistic > 0.1 and p-value < 0.01). Briefly, filtered datasets whose distribution of cytosines at mapped locations is significantly lower than unfiltered datasets have one read removed and are re-tested for differences in their distribution. If the distributions are more similar (as measured through the same criteria), it is filtered out. This process is repeated until distributions are no longer significantly different or if all reads are removed. Metagenomic abundances of filtered datasets are estimated using GRAMMy (version 1)17. Microbial abundance in downstream analyses was quantified as Molecules Per Million reads (MPM).

Total RNA isolation

In the centrifugation method, urinary cell lysates were prepared by centrifugation of whole urine at 2000g for 30 min and harvesting the cell pellets followed by cell lysis with 350 μl of Buffer RLT (Qiagen, Cat# 79216) + β-mercaptoethanol. In the filtration method, urine was pushed through a Whatman 1.6 μm GF/A filter and urinary cell lysate was collected by pushing 700 μl of Urine RNA Buffer (Zymo Research, Cat# R1038-2-50). Total RNA was extracted from both lysates with RNeasy Mini Kit (Qiagen, Cat 74104). The purity of isolated total RNA (A260/280) and the amount of RNA were measured using the Nanodrop One spectrophotometer. Nanodrop replicates were within 0.2 for the A260/280 ratio and 1 ng/μl for the concentration.

Reverse transcription of total RNA to cDNA

Total RNA was reversed transcribed to cDNA using TaqMan Reverse Transcription Kit (Applied Biosystems, Cat N8080234). To normalize the different total RNA yields from the samples, total RNA was converted to cDNA at a concentration of 1 μg total RNA in 100 μl volume. The reaction contained 1 × TaqMan reverse transcription buffer, 500 μM each of 4 dNTPs, 2.5 μM of Random Hexamer, 0.4 Unit/μl of RNase inhibitor, 1.25 Unit/μl of MultiScribe Reverse Transcriptase and 5.5 mM of Magnesium Chloride, and was heated at 25 °C for 10 min, 48 °C for 30 min, and 95 °C for 5 min.

Preamplification enhanced real time PCR quantification

Each cDNA was pre-amplified with custom-designed primer pairs using Platinum® Multiplex PCR Master Mix (Applied Biosystems, Cat 4464268). Each reaction contained 5.0 μl Platinum® Multiplex PCR Master Mix, 3.0 μl cDNA, 1.68 μl primer mix (50 μM sense and 50 μM antisense primers for each gene), and 0.32 μl RNase/DNase free water. The reaction was heated at 95 °C for 2 min, followed by 11 cycles of 95 °C for 30 s, 60 °C for 90 s, and 72 °C for 1 min, and held for 72 °C for 10 min. The 10 μl of pre-amplified cDNA is then diluted in 290 μl TE to use in the absolute quantification of mRNAs performed on the QuantSudio™ 6 Flex Real Time PCR system (Applied Biosystems).

Statistical analysis

All statistical methods for cfDNA analysis were performed in R version 4.0.5. Groups were compared using two-sided Wilcoxon Rank Sum and Kolmogorov–Smirnov tests. The correlation between the Ct values obtained in the quantitative PCR (qPCR) assays following total RNA extraction using the centrifugation and filtration method were compared using Pearson’s method. Boxplots with 10th, 25th, 50th, 75th and 90th percentile of the RNA parameters, concentration, amount and A260/280 ratios, as well as the Ct values obtained in the quantitative PCR (qPCR) assays were created. Boxes in the boxplots indicate the 25th and 75th percentile, the band inside the box indicates the median value and the whiskers extend to 10th and the 90th percentile. The paired Ct values associated with each mRNA measurements were compared using the Wilcoxon signed-rank test, followed by a Bonferroni correction method to adjust for type I error. P values are reported for those that remained significant after Bonferroni correction and ‘ns’ is reported if the p values were not significant after applying the Bonferroni correction.