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StartMicrobiologyThe standardisation of the method to metagenomic human intestine evaluation: from pattern...

The standardisation of the method to metagenomic human intestine evaluation: from pattern assortment to microbiome profiling


DNA extraction and NGS library development

The DNA extraction process was examined on the three volunteer samples (S1, S2, S3) and the BL pattern. For particulars on volunteers’ samples and BL pattern preparation, see Strategies and Samples sections. Every of the S1, S2, S3 and BL samples was processed in triplicate. As offered in Fig. 1, three technical repetitions differed solely within the time of mechanical pattern homogenisation (5, 10 or 15 min). Because of this, 12 DNA extracts had been obtained. From a single stool pattern (250 mg), 2–20 µg of DNA was extracted. Excessive-quality genomic DNA with a molecular weight of roughly 20 kb was obtained from all samples (Supplementary Fig. 2). The standard of DNA was impartial of the time of pattern homogenisation, and solely minor variations within the quantity of the remoted DNA when it comes to homogenisation time had been noticed. For the S1 and S2 samples, the extension of the homogenisation time resulted in a slight discount within the quantity of extracted DNA from the stool samples, whereas for the S3 samples, the quantity of remoted DNA was steady, whatever the homogenisation time. For the BL pattern, the longest homogenisation time resulted in a slight enhance within the quantity of remoted DNA (Supplementary Desk 4). To confirm the correctness and effectivity of DNA extraction, we used the GD pattern, which was a commercially accessible genomic DNA isolate from ATCC bacterial combination.

Every package produced libraries of various quantities and measurement distributions (for particulars, see Supplementary File 1). KAPA libraries had the longest fragments, which can counsel inadequate enzymatic fragmentation on the preliminary step of library preparation. Furthermore, KAPA libraries had essentially the most variable common fragment measurement (min 550 bp, max 1862 bp), library focus (min 4.8 ng/µL, max 118 ng/µL) and, consequently, the ultimate quantity of library DNA (min 96.8 ng, max 2360 ng). With the Qiagen package, the very best quantities of libraries had been obtained (870–1730 ng, imply 1372.7 ng), which signifies very environment friendly amplification, however the next variety of PCR duplicates might be an undesirable consequence. Nextera libraries mirrored essentially the most homogeneous and repeatable outcomes, confirming the exactly optimised fragmentation (opposite to the KAPA and Qiagen kits, which utilise enzymatic DNA fragmentation, Nextera applied the tagmentation technique) and amplification circumstances within the Illumina protocol.

Management of sequencing high quality

To regulate the standard of the ensuing sequencing reads, we first in contrast how the variety of reads was distributed throughout the libraries ready with specific kits. KAPA gave essentially the most numerous outcomes with the bottom variety of reads (16,790,404.8, std. 9,219,148.96), whereas Qiagen resulted within the highest variety of reads (31,589,315.57, std. 7,735,638.94) but in addition larger variability than Nextera (20,542,238.62, std. 1,085,110.33) (Fig. 2A, Supplementary Desk 6), which is according to our measurements of the ready libraries. When it comes to these standards, we discovered Nextera to be compromise between the variety of reads and reproducibility.

Determine 2
figure 2

Variety of reads generated for every DNA pattern processed with KAPA, Nextera and Qiagen library preparation kits, following totally different homogenisation instances: 0 min for GD combine, 10, 15 and 20 min for actual samples (S1, S2, S3) and BL (A). The variety of reads undetermined throughout demultiplexing was included within the plot as pattern U. Variety of reads with undetermined barcodes that weren’t efficiently demultiplexed for every of the kits (B).

We additionally recognized 39,651,338 reads with undetermined barcodes. We analysed the highest undetermined barcodes for every package and located that there was a major hole between the variety of undetermined reads originating from the KAPA package and different kits (8,973,700 vs 1,476,300). A lot of the undetermined barcodes had a single i5 index coming from Illumina’s TruSeq Common Adapter sequence (Fig. 2B, Supplementary Desk 7). Throughout learn preprocessing, we trimmed the adapter sequences and poly-G and filtered out reads shorter than 140 bases to take away bias in taxonomy profiling. The variety of reads maintained after this remedy might be present in Supplementary Desk 6.

Establishing taxonomy profiling process

For the following step, we wished to decide on and calibrate a bioinformatics process for profiling the microbial taxonomy. We have now taken under consideration the 2 main instruments which can be used for taxonomy profiling: MetaPhlAn2 and Kraken2 with Bracken correction, which use orthogonal approaches to profiling the composition of the metagenomic pattern. Initially, we examined each instruments on the in silico samples, representing ATCC communities with an excellent (ISE) and staggered (ISS) distribution of species.

We noticed that MetaPhlAn2 performs worse when it comes to figuring out species (recall 91.67% for even abundance and 66.67% for staggered species abundance) than the Kraken2/Bracken mixture with a default confidence threshold of 0.0 (recall 100% for each even and staggered species abundance) (Fig. 3, Supplementary Desk 8). The fraction of the appropriately recognized species within the recovered taxonomic profiles was larger for MetaPhlAn2 than for Kraken2/Bracken mixture (78.57% vs 22.64% for ISS and 80% vs 64.71% for ISE, respectively). Nevertheless, weighted precision for MetaPhlAn2 was a lot decrease than for Kraken2/Bracken (34.03% for ISE and 41.89% for ISS with MetaPlAn2 vs 97.65% and 99.22% with Kraken2’s default confidence threshold). Furthermore, for each samples, the RMSE of anticipated versus noticed abundance in appropriately recognized species was a lot larger for MetaPhlAn2 profiling than for Kraken2/Bracken mixture in default runs, with RMSE ranges of 4.85 and 0.39, respectively, within the ISE pattern with even species distribution, presenting an excellent bigger distinction for the ISS pattern with a extra numerous species distribution (RMSE 11.34 for MetaPhlAn2 and 0.2 for Kraken2/Bracken).

Determine 3
figure 3

Group profiling metrics for the ISE pattern (in silico even abundance) (A) and for the ISS pattern (in silico staggered abundance) (B). Values for default settings of taxonomy profiling instruments are highlighted in purple. The Kraken2/Bracken mixture recognized all anticipated species, versus MetaPhlAn2. Numerous Kraken2 confidence threshold values had been examined to succeed in the next weighted precision degree and decrease RMSE.

We tried to cut back the fraction of incorrectly detected species and enhance the potential of Kraken2/Bracken in group reconstruction. For this function, we modified the boldness threshold parameter, which is supposed to enhance the constancy of group reconstruction. Subsequently, we profiled the samples with Kraken2 and Bracken utilizing totally different confidence threshold values, specifically, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6. This parameter is anticipated to cut back the variety of false constructive outcomes by establishing a minimal proportion of okay-mers of a given learn which were mapped to the assigned species. An abundance abstract for the default Kraken2/Bracken and MetaPhlAn2 parameters, in addition to for various Kraken2 confidence thresholds, might be present in Supplementary Desk 9. Outcomes include many species, the vast majority of which shouldn’t be detected within the synthetic samples. Of discover, the species that weren’t anticipated had been detected in a negligible abundance and solely below one or two examined settings of confidence thresholds. These species must be handled as artifacts and, when contemplating taxonomy and its which means, must be filtered out from the outcomes.

Since MetaPhlAn2 was not capable of detect all of the anticipated species, exhibiting notably poor recall for the ISS pattern, which higher represents the abundance distribution anticipated in the actual samples, we determined to make use of Kraken2/Bracken additional within the taxonomy profiling process by tweaking the boldness threshold parameter. Growing the brink to 0.1 already introduced notable enchancment when it comes to false species discovery, whereas additional rising this confidence threshold gave a slight enhance in abundance error (RMSE) within the ISE pattern. We determined to proceed additional with 0.1 within the processing of actual samples to keep away from introducing further bias in pattern composition.

Human DNA content material evaluation

Learn mapping (BWA MEM, default parameters, seed size of 19) towards the human genome (hg38) yielded an unexpectedly excessive variety of mapped reads (common human learn fraction per pattern 23.36%, commonplace deviation 10.38) (Fig. 4A). Curiously, we observed the identical for the ISE and ISS in silico samples, which had been generated based mostly on solely bacterial genomes. Detailed evaluation revealed that the overwhelming majority of reads that mapped to the human genome had been acknowledged by Kraken2 as of bacterial origin, which suggests “false” mappings by BWA. Subsequently, we targeted on the distribution of the entire variety of bases alongside the learn that mapped to the hg38 reference within the ATCC bacterial combine pattern (N1BL pattern spiked with human PBM cells).

Determine 4
figure 4

Fractions of reads mapped to the human genome in BL (A) with default (19 nt) and elevated (26 nt) minimal BWA MEM seed size and (B) donor samples (minimal BWA MEM seed size 26 nt) for various kits and homogenisation instances.

We noticed two teams of mapped reads—one similar to reads mapping to the reference with a brief overlap (3,139,174 reads with lower than 23 nucleotides overlapping with the reference) and a big peak of 509,219 reads mapping to the reference on all the learn size. We anticipated that the reads with a brief overlap and the reference fragment would have originated from comparable areas shared by the human and bacterial species (Fig. 5A). Visualizing the mapping size distribution over the learn, we additionally noticed two peaks within the N1BL pattern. One peak corresponded to actual human reads, the place mapped areas lined almost the complete learn size, whereas the opposite peak, characterised by the low mapping size, noticed areas of unintended sequence similarity. We discuss with the latter reads as false positives, as they incorrectly contributed to the noticed fraction of human materials within the samples.

Determine 5
figure 5

Distribution of the variety of matches alongside the reads mapped to the human genome and corresponding mapping high quality with default (A,C—19 nt) and elevated BWA minimal seed size parameters (B,D—26 nt).

To eradicate the noise coming from false positives, we elevated the minimal seed size threshold for BWA to the ninety fifth percentile of false positives’ mapping seed size (from 19 to 26 bases) (Fig. 5A,B). Growing this threshold eradicated multimapping reads of low high quality, now indicating two clear peaks similar to reads of a excessive chance of mapping appropriately (MAPQ = 60) and people mapping to a number of areas equally properly (MAPQ = 0) (Fig. 5C,D).

After rising the brink in BWA, we noticed that for not one of the BL samples, the human learn content material exceeded the anticipated 3% (Fig. 4A). For the KAPA and Nextera kits, the fraction of human reads within the samples barely decreased with rising homogenisation time, starting from 2.83% for K1BL to 2.13% for K3BL and a pair of.88% for N1BL to 2.53% for N3BL, the place 1/2/3 corresponds to 10/15/20 min of homogenisation time. For QIAseq, we noticed a barely larger fraction of human reads (2.95% for Q1BL to three.11% for Q3BL), with Q2BL exhibiting the bottom human learn content material of two.6%.

Extra variations between isolation kits had been noticed within the outcomes of mapping reads towards the human genome from donor samples S1, S2 and S3 (Fig. 4B). Amongst all three kits, QIAseq gave the very best variety of human reads (avg. 0.99%, sd. 0.61%) compared to KAPA (avg. 0.04%, sd. 0.03) and Nextera (avg. 0.1%, sd. 0.01). The influence of the library preparation package was additional supported by the outcomes of PERMANOVA, by which we assessed the influence of the isolation package and homogenisation time on the human reads fraction. The check confirmed that kits performed a major position within the fraction of reads mapped to the human genome (F worth 74.012, R2 0.866, p worth 0.001), whereas neither homogenisation time (F worth 0.916, R2 0.012, p worth 0.395) nor the mixture of homogenisation time and package (F worth 0.787, R2 0.018, p worth 0.577) was vital (Desk 2).

Desk 2 Outcomes of PERMANOVA check on the influence of homogenisation time and library prep package on the variety of human reads.

We additionally checked how the prefiltering of human reads impacts bacterial group profiling with Kraken2. We calculated recall, weighted precision and RMSE for GD and BL samples with out filtering and with prefiltering of human reads with BWA with a seed size of 19 and 26 nucleotides (Fig. 6). Although the outcomes of mapping confirmed that BWA with a seed size of 19 provides a excessive quantity of FP (reads categorised as human species that in actuality are of bacterial origin), our evaluation revealed that prefiltering with decrease (19) or larger (26) seed size doesn’t have an effect on the bacterial group profiling, giving comparable outcomes to nonfiltered information. Though the weighted precision for unfiltered BL information was decrease than that of filtered BL information (97.44% vs > 99.9%), it have to be famous that weighted precision metrics for unfiltered information didn’t bear in mind reads categorised as Homo sapiens species as TP. Furthermore, as anticipated, the abundance of reads from Homo sapiens was estimated to be 2–3% of BL samples; the weighted precision for filtered and unfiltered information was comparable.

Determine 6
figure 6

Bacterial group profiling metrics for the BL samples with out filtering human reads (A) and with prefiltered human reads (BWA, seed size of 19 (B) and 26 (C)). Weighted precision metrics in (A) don’t bear in mind reads categorised as Homo sapiens species as TP (anticipated abundance of reads from Homo sapiens was estimated to be 2–3% of a pattern). 1/2/3 corresponds to 10/15/20 min of homogenisation time.

Group reconstruction evaluation for GD and BL samples

The outcomes of the group reconstruction evaluation with Kraken2 for GD and BL samples might be present in Supplementary Desk 8 (GD samples) and Supplementary Desk 9 (BL samples). For each GD and BL samples, all the anticipated species had been recognized by Kraken2—the recall parameter was equal to 100% for all of the samples and examined circumstances. The variety of false discoveries was additionally low for each GD and BL samples and circumstances (weighted precision exceeding 99.4% for all of the samples and circumstances), with barely larger values obtained for BL samples (Fig. 6C, Supplementary Desk 10). Nevertheless, we did observe deviation from the anticipated species proportions, which was mirrored within the RMSE of abundance, with the K3BL pattern barely deviating from the opposite samples (RMSE 2.85). General, GD confirmed a decrease RMSE of appropriately detected species abundance than BL (highest worth 1.97 for NGD and lowest worth 2.45 for K1BL). This was even supposing the GD pattern composition was extra taxonomically advanced, with larger species variety and staggered abundance. This remark was additionally supported by the similarity evaluation. We noticed the next degree of similarity to the unique composition for GD than for BL; the very best Bray–Curtis dissimilarity worth amongst GD was 0.17 (NGD, Fig. 7A, Supplementary Desk 11), whereas the bottom dissimilarity of BL was 0.22 (K2BL, Fig. 7B). These observations level to the bias launched by the isolation step in BL samples. The GDQ pattern ready with QIAseq was essentially the most much like the anticipated group, with a Bray–Curtis metric of 0.13 and RMSE of 1.57 (Fig. 7A, Supplementary Tables 12 and 13).

Determine 7
figure 7

Bray–Curtis beta variety clustering of GD (A) and BL (B) samples. Genomic DNA samples (GD) confirmed decrease general dissimilarity from the anticipated species composition than whole-cell isolates (BL). BL samples ready with the identical homogenisation instances confirmed decrease between-sample variety. “Anticipated” refers back to the identified composition of samples specified by the producer. 1/2/3 within the pattern title corresponds to 10/15/20 min of homogenisation time.

Clustering of the Bray–Curtis dissimilarity revealed that BL samples clustered by the homogenisation time moderately than the library preparation package (Fig. 7B, Supplementary Desk 13). Samples homogenised for 20 min fashioned one cluster (common Bray–Curtis for K3BL/N3BL/Q3BL cluster was 0.079), and samples homogenised for 15 min fashioned a second cluster (common Bray–Curtis for K2BL/N2BL/Q2BL samples was 0.153). Though clustering revealed the Q1BL pattern as a possible outlier, because it clustered individually from all the opposite samples on account of a low beta-diversity, two different samples homogenised for 10 min, K1BL and N1BL, fashioned a strong cluster. Pattern N1BL confirmed to be the closest to the anticipated composition among the many BL samples, with beta-diversity of N1BL versus Anticipated of 0.219.

Actual samples profiling

Versus the GD and BL mock samples, it was not doable to match volunteers’ samples S1, S2, and S3 to any reference composition. To analyze the influence of isolation kits and homogenisation time on intestine microbiome profiling, we calculated Bray–Curtis dissimilarity between samples coming from the identical donor ready with every of the protocols (Fig. 8, Supplementary Desk 13). We noticed that for every of the S1 and S2 samples, 10 min of homogenisation resulted in additional distinct communities than these noticed between 15 and 20 min of homogenisation. This remark was much less distinct for the S3 pattern.

Determine 8
figure 8

Bray–Curtis dissimilarity of donor samples S1 (A), S2 (B), and S3 (C) ready with totally different kits and homogenisation instances. Greater similarity was discovered between samples homogenised for 10 min than for 15- and 20-min homogenisation in S1 (A) and S2 (B) samples. For S3 (C), samples confirmed a low degree of dissimilarity, with 10- and 15-min homogenisation clustering collectively throughout the similar library preparation package. 1/2/3 corresponds to 10/15/20 min of homogenisation time.

Furthermore, whereas the S2 pattern confirmed clear clusters of samples remoted with the identical homogenisation time, for 2 different samples, we noticed that after the homogenisation time was elevated from 10 to fifteen and 20 min, samples tended to cluster into small teams by the isolation kits. For S1, we noticed a definite cluster of KAPA samples (K2S1, K3S1) and a cluster of Nextera and QIAseq, additional cut up by homogenisation time (N2S1 with Q2S1 and N3S1 with Q3S1). For S3, we noticed distinct clusters of N2S3 + N3S3 and Q2S3 + Q3S3 and K2S3 + K3S3. Taxonomic profiles obtained for volunteer samples might be present in Supplementary Desk 14, and the taxonomical tree for the Micro organism kingdom might be present in Supplementary Figs. 3, 4 and 5 for samples S1, S2 and S3, respectively. There are some variations seen inside kits and homogenisation time for a given pattern (Supplementary Figs. 6, 7, 8, 9, 10 and 11). Outcomes of pairwise comparisons between totally different kits and totally different homogenisation instances for every pattern (S1/S2/S3) for Micro organism kingdom clades might be present in Supplementary Desk 15.

Gram-positive and gram-negative species isolation in BL samples

We assessed gram-positive/gram-negative species isolation bias in BL samples, in addition to in volunteers’ samples. Moreover, we checked the gram-positive/gram-negative species’ abundance ratio within the GD samples that originated from a genomic DNA combine (Fig. 9A). Our evaluation confirmed that general, the proportion of gram-positive to gram-negative species’ abundance in GD samples was decrease than anticipated. Since these samples weren’t subjected to isolation, we attribute the final variations to the distinction within the genomes’ size (common genome size 2,536,718 bp for gram-positive and 4,282,149 bp for gram-negative species), which can have resulted in proportionally much less gram-positive species’ genomes captured by the reads. Since gram-positive species had decrease GC content material than gram-negative species’ genomes (common 36.24% and 49.57%, respectively), we dominated out an amplification step as a possible supply of variations within the species’ abundance. Furthermore, we noticed the Nextera package to present outcomes barely extra distant from the anticipated than KAPA and Qiagen kits. The biggest variations in abundance between KGD and NGD had been current for Escherichia coli (gram-negative species, 0.183% in KGD vs 0.211% in NGD) and Staphylococcus epidermidis (gram-positive species, 0.187% in KGD vs 0.160% in NGD). For QGD and NGD, the biggest noticed distinction in abundance was for Rhodobacter sphaeroides (gram-negative species, 0.265% and 0.231% abundance, respectively).

Determine 9
figure 9

Anticipated versus noticed proportion of gram-positive to gram-negative species in management pattern GD (A) and remoted BL samples (B). For BL, we noticed the next fraction of gram-positive species than within the authentic bacterial combine, and the proportion of gram-positive micro organism elevated with longer homogenisation instances.

Within the BL bacterial combine with an excellent species abundance distribution, we anticipated to see twice as few gram-positive species as gram-negative species (gram-positive/gram-negative ratio 0.5). Nevertheless, for all of the kits, versus the GD samples, we noticed the next fraction of gram-positive species than anticipated (avg. gram-positive/gram-negative ratio 0.885), which persistently elevated with homogenisation time (Fig. 9B). We additional in contrast the noticed abundance of each gram-positive and gram-negative species with abundances reported by ATCC producer38 (Supplementary Desk 1) for ATCC® MSA-2006™ to research whether or not the bias in species abundance that we noticed was according to these outcomes, which resulted in a gram-positive/gram-negative species ratio of 0.657. Nonetheless, for each gram-positive and gram-negative species, we noticed a linear correlation of abundances, indicating a constructive relationship between our abundance and the producer’s abundance (Fig. 10). Correlation coefficients for gram-negative species ranged between 0.71 in K3BL and 0.82 in Q3BL, whereas correlation coefficients for gram-positive species ranged between 0.72 in K3BL and 1 in K2BL.

Determine 10
figure 10

Species abundance noticed for ATCC® MSA-2006™ versus our noticed abundances, labelled by Gram stain standing. For each gram-positive and gram-negative species, our outcomes correlate with the producer’s findings; nonetheless, for gram-positive species, the correlation was not vital in all of the samples, as there have been solely 4 g-positive species in ATCC® MSA-2006™.

Whereas zooming in to the deviations from the anticipated abundance, which was 8.3% for all of the species within the BL pattern, we noticed teams of species that had been systematically both over- or underrepresented (Supplementary Fig. 12). Amongst species with larger abundance than anticipated, we recognized Bifidobacterium adolescentis (median abundance 13.24%), Clostridioides difficile (median abundance 14.97%) and Lactobacillus plantarum (median abundance 11.62%). Underrepresented species included Escherichia coli (median abundance 4.04%), Helicobacter pylori (median abundance 2.33%), Fusobacterium nucleatum (0.96%) and Salmonella enterica (4.66%). Whereas 2 out of three overrepresented species belonged to the gram-positive group of micro organism (specifically B. adolescentis and C. difficile), all of the underrepresented species belonged to the gram-negative group.

We investigated the doable influence of Gram staining standing, genome size and GC content material on the median abundance of the species throughout BL samples. We match a linear mannequin with a top-down method, which confirmed that in our information, solely the Gram staining standing is a major predictor of median abundance, with the coefficient of 0.0634 and a p worth of 0.0156 for the ultimate mannequin, which included Gram staining standing solely (Supplementary File 2).

Gram-positive and gram-negative species isolation effectivity

We noticed mapped reads for Zymo-spikes, not solely in spiked volunteer samples S1, S2 and S3, the place the Zymo management was added but in addition in BL and GD samples with out spikes (Supplementary Desk 16). Nevertheless, the general fraction of those Zymo-spikes’ genomes lined was a lot decrease for BL and GD samples (1.08–1.81% for Allobacillus halotolerans and 0.31–0.42% for Imtechella halotolerans) than for volunteer samples (5.65–43.06% for Allobacillus halotolerans and seven.36–73.06% for Imtechella halotolerans). Moreover, the median protection of these areas was a lot larger in BL and GD (ranging between 21 × and 207 × for Allobacillus halotolerans and 17 × to 227 × for Imtechella halotolerans) than in volunteer samples (median protection of 1 × for Allobacillus halotolerans and 1–2 × for Imtechella halotolerans). This means that reads mapping to Zymo-spikes in BL and GD originate from a slender vary of extremely comparable areas throughout species, whereas in volunteer samples, there’s low however constant protection alongside the anticipated genomes from spikes. Nonetheless, areas comparable throughout the entire microbial group would influence mappings within the volunteer samples; subsequently, inferring gram-positive/gram-negative species isolation bias required evaluation of reads’ protection moderately than immediately evaluating a fraction of mapped reads.

Having a median protection of 1 × throughout many of the genomic sequence for the volunteer samples, the proxy for pressure abundance was a fraction of the genome that was noticed in sequencing reads. The ratio of the genome positions that had been lined by reads throughout mapping to the reference genomes of Allobacillus halotolerans and Imtechella halotolerans means that in our experiment, a slight bias towards extra environment friendly isolation of gram-negative species could possibly be noticed for actual samples (25.47% of the Allobacillus halotolerans genome lined in comparison with 42.50% for Imtechella halotolerans). This remark was supported by the Wilcoxon check, leading to a p worth of 1.9e−04. Nevertheless, in volunteer samples, we didn’t observe a major influence of homogenisation time, package or mixture of each on the Allobacillus halotolerans/Imtechella halotolerans ratio, as supported by PERMANOVA (Desk 3).

Desk 3 Outcomes of PERMANOVA check on influence of homogenisation time and library prep package on gram-positive/gram-negative species isolation bias (Allobacillus halotolerans/Imtechella halotolerans ratio).
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