Cell-Free Microbial DNA Analysis: Effects of Blood Plasma and Serum Quantity, Biobanking Protocols, and Isolation Kits.
Author(s): Nikitina D, Lukosevicius R, Tilinde D, Muskieta T, Hov JR, Melum E, Klovins J, Org E, Kiudelis G, Kupcinskas J, Skieceviciene J
Publication: Biopreserv Biobank, 2024, Vol. , Page
PubMed ID: 38416864 PubMed Review Paper? No
Purpose of Paper
This paper compared the next generation sequencing (NGS) profiles of cell-free microbial DNA that was extracted using three different kits from different volumes (100, 200 or 500 µL) of single- or double-spun sodium citrate plasma, K2EDTA plasma, or serum.
Conclusion of Paper
Ward’s hierarchical clustering and principal component analysis clustered all specimens that were extracted using the Norgen kit distinctly from those that were extracted using the other two kits; samples were not segregation based on the number of centrifugation steps, collection tube type, or plasma/serum aliquot volume. In 52 of 54 (96%) cases, PERMANOVA identified significant differences in bacterial sequence abundance based on extraction method, with all comparisons showing differences between Norgen and the other two kits. In contrast, only 9% of specimens displayed differences based on specimen volume and 4% based on collection tube type. DNA extraction method had the largest effect on the bacterial composition with more similarity observed between the Qiagen and MagMAX methods at both the phyla and genus level than between either kit and the Norgen kit. Sixteen of the 20 most abundant phyla were common between the Qiagen and MagMAX extraction methods, but only 5 of these were found among the top 20 when extraction was with the Norgen kit. Compared to specimens extracted using the Qiagen or MagMAX kit, specimens extracted using the Norgen kit had significant differences in the levels of 548 and 533 bacteria, respectively (reflecting approximately 30 bacteria per group). Only 25 bacteria were found to have significantly different levels between Qiagen and MagMAX kits. By comparison, collection tube type, the number of centrifugation steps, and plasma/serum aliquot volume had an impact on the β-diversity of 28, 23, and 27 bacteria, respectively, and many of these were only observed when Norgen was used for extraction. Collection tube type affected Pielou’s evenness but only when serum/plasma was separated by single- spin centrifugation and extraction was from 500 µL of plasma/serum using the MagMAX kit. The volume of specimen that was used as input did not affect bacterial diversity, but bacterial richness was lower when extraction was from 100 µL EDTA plasma using MagMAX or 100 µL sodium citrate plasma using Qiagen or Norgen kits compared to at least one of the larger volumes (200 or 500 µL). Bacterial richness was higher when plasma/serum was obtained by double rather than single centrifugation when extraction was with the Norgen kit, regardless of collection tube type. Bacterial richness was generally higher when extraction was with the Norgen kit than the other kits evaluated, with significant differences found in the double centrifugation group (regardless of volume or tube type) and when extraction was from 100 or 500 µL serum.
Studies
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Study Purpose
This study compared the next generation sequencing (NGS) of cell-free microbial DNA that was extracted using three different kits from different volumes (100, 200, or 500 µL) of single or double spun sodium citrate plasma, K2EDTA plasma, or serum. Blood was collected from four healthy volunteers (2 men and 2 women) into BD Vacutainer K2EDTA tubes, BD Vacutainer sodium citrate tubes, and BD Vacutainer serum gel tubes. Tubes were stored at room temperature for approximately 30 min before serum/plasma separation. Serum/plasma were obtained by a single centrifugation at 1500 g for 15 minutes or by dual centrifugation by two spins at 1500 g for 15 min. Plasma/serum were aliquoted (100 µL, 200 µL, 500 µL) and frozen at -80°C for 1 month. DNA was extracted from the serum/plasma aliquots using the Norgen Plasma/Serum Cell-fee Circulating DNA Purification Micro Kit, the MagMAX Cell-free DNA Kit and the QIAamp MinElute Cell-free Circulating DNA Mini kit. 16S rRNA libraries were prepared using an in-house protocol and were sequenced using the MiSeq Reagent Kit v3. Bacterial sequences were assigned taxonomic affiliation and phyla; sequences with <1000 counts were excluded as were sequencing variants that were detected in <3 specimens (of the 216 sequenced). Differences between processing groups were analyzed using PERMANOVA (9999 permutations). The “Bacterial richness, Pielou’s evenness, and Simpson’s and Shannon’s diversity indices” were used to analyze α-diversity. Differences were significant when the Benjamini–Hochberg FDR corrected P-value (q-value) was <0.05.
Summary of Findings:
Ward’s hierarchical clustering method clustered all specimens extracted using the Norgen kit distinctly from those extracted using the other two kits, with considerable overlap observed between the Qiagen and MagMAX methods. Within the extraction method clusters, specimens did not clearly segregate by the number of centrifugation steps, collection tube type, or plasma/serum aliquot volume. The main genus driving the separation based on extraction method were Gordonia, Micrococcus, Streptococcus, Delftia, and Pseudomonas. Simarly, principal component analysis also separated specimens extracted using the Norgen kit from those extracted using the other two kits, with no separation based on the number of centrifugation steps, collection tube type, or plasma/serum aliquot volume observed. In 52 of 54 cases (96%), PERMANOVA identified significant differences in microbial sequence abundance based on extraction method, regardless of number of centrifugation steps, collection tube type, or plasma/serum aliquot volume. As expected, differences were significant between Norgen and the other two methods under all conditions, but significant differences between Qiagen and MagMAX methods were found with two exceptions (using 100 or 500 µL single-spun serum or 200 µL single spun sodium citrate plasma). Only 9% of specimens displayed differences based on aliquot volume and 4% based on tube type. DNA extraction method had the largest effect on the bacterial composition, with more similarity observed between the Qiagen and MagMAX extraction methods at both the phyla and genus level than between either kit and the Norgen kit; 16 of the 20 most abundant phyla were common among the Qiagen and MagMAX methods, but only 5 of these were found among the top 20 when extraction was with the Norgen kit. Compared to specimens extracted using the Qiagen or MagMAX kit, specimens extracted using the Norgen kit had significant differences in levels of 548 and 533 bacteria, respectively (30 bacteria per group). Only 25 bacterial abundances were found to be significantly different between Qiagen and MagMAX kits. By comparison tube type, the number of centrifugation steps, and plasma/serum aliquot volume had an impact on the β-diversity of 28, 23, and 27 bacteria, respectively, and many of these were only observed when the Norgen method was used for extraction. Limited effects of collection tube type, input volume, and extraction method on α-diversity were observed. Collection tube type affected Pielou’s evenness but only when serum/plasma was separated by single centrifugation and extraction was using 500 µL of Serum/Plasma along with the MagMAX kit (P=0.042). The volume of input did not affect bacterial diversity, but bacterial richness was lower when extraction was from 100 µL EDTA plasma using MagMAX kit or 100 µL sodium citrate plasma using the Qiagen or Norgen kits compared to at least one larger volume (200 or 500 µL, P<0.05). Bacterial richness was higher when plasma/serum was obtained by double rather than single centrifugation and when extraction was with the Norgen kit, regardless of collection tube. Bacterial richness was generally higher when extraction was with the Norgen kit compared to the other kits evaluated, with significant differences found in the double centrifugation group (regardless of volume or tube type), and when extraction was from 100 or 500 µL serum.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution K2EDTA plasma tube
Sodium citrate plasma tube
Serum gel tube
Analyte Extraction and Purification Analyte isolation method Norgen plasma/serum cell-free circulating DNA purification micro kit
MagMAX cell-free DNA
QIAamp MinElute cell-free circulating DNA mini kit
Biospecimen Aliquots and Components Aliquot size/volume 100 µL
200 µL
500 µL
Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum