| 1. |
- Hasmats, Johanna, et al.
(författare)
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Assessment of Whole Genome Amplification for Sequence Capture and Massively Parallel Sequencing
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:1, s. e84785-
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Tidskriftsartikel (refereegranskat)abstract
- Exome sequence capture and massively parallel sequencing can be combined to achieve inexpensive and rapid global analyses of the functional sections of the genome. The difficulties of working with relatively small quantities of genetic material, as may be necessary when sharing tumor biopsies between collaborators for instance, can be overcome using whole genome amplification. However, the potential drawbacks of using a whole genome amplification technology based on random primers in combination with sequence capture followed by massively parallel sequencing have not yet been examined in detail, especially in the context of mutation discovery in tumor material. In this work, we compare mutations detected in sequence data for unamplified DNA, whole genome amplified DNA, and RNA originating from the same tumor tissue samples from 16 patients diagnosed with non-small cell lung cancer. The results obtained provide a comprehensive overview of the merits of these techniques for mutation analysis. We evaluated the identified genetic variants, and found that most (74%) of them were observed in both the amplified and the unamplified sequence data. Eighty-nine percent of the variations found by WGA were shared with unamplified DNA. We demonstrate a strategy for avoiding allelic bias by including RNA-sequencing information.
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| 2. |
- Hu, Yue O. O., et al.
(författare)
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Stationary and portable sequencing-based approaches for tracing wastewater contamination in urban stormwater systems
- 2018
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Urban sewer systems consist of wastewater and stormwater sewers, of which only wastewater is processed before being discharged. Occasionally, misconnections or damages in the network occur, resulting in untreated wastewater entering natural water bodies via the stormwater system. Cultivation of faecal indicator bacteria (e.g. Escherichia coli; E. coli) is the current standard for tracing wastewater contamination. This method is cheap but has limited specificity and mobility. Here, we compared the E. coli culturing approach with two sequencing-based methodologies (Illumina MiSeq 16S rRNA gene amplicon sequencing and Oxford Nanopore MinION shotgun metagenomic sequencing), analysing 73 stormwater samples collected in Stockholm. High correlations were obtained between E. coli culturing counts and frequencies of human gut microbiome amplicon sequences, indicating E. coli is indeed a good indicator of faecal contamination. However, the amplicon data further holds information on contamination source or alternatively how much time has elapsed since the faecal matter has entered the system. Shotgun metagenomic sequencing on a subset of the samples using a portable real-time sequencer, MinION, correlated well with the amplicon sequencing data. This study demonstrates the use of DNA sequencing to detect human faecal contamination in stormwater systems and the potential of tracing faecal contamination directly in the field.
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| 3. |
- Hu, Yue, 1987-, et al.
(författare)
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Stationary and portable sequencing-based approaches for tracing wastewater contamination in urban stormwater systems
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Urban sewer systems consist of wastewater and stormwater sewers, of which typically onlythe wastewater is processed before being discharged. Occasionally, misconnections ordamages in the network occur, resulting in wastewater entering the stormwater system andbeing discharged without prior processing. Cultivation of faecal indicator bacteria, such asEscherichia coli (E. coli), is the current standard for tracing wastewater contamination. Thismethod is cheap but cannot be employed in the field and is characterised by its limitedspecificity. Here, we compared the E. coli culturing approach with two different DNAsequencing-based methodologies (i.e., 16S rRNA amplicon sequencing on the IlluminaMiSeq platform and shotgun metagenomic sequencing on an Oxford Nanopore MinIOndevice), analysing 73 stormwater samples collected throughout the Stockholm city areas.High correlations were obtained between E. coli culturing counts and frequencies of humangut microbiome sequencing reads (via amplicon sequencing), indicating that E. coli is indeeda good indicator of faecal contamination. In contrast to E.coli culturing, amplicon sequencingcould, however, further distinguish between two different sources of contamination in anarea, where misconnections in the stormwater system were later on detected. Shotgunmetagenomic sequencing on a subset of the samples using the portable Oxford NanoporeMinION real-time sequencing device correlated well with the amplicon sequencing data. Insummary, this study shows that DNA sequencing allows distinguishing differentcontamination sources in stormwater systems and demonstrates the potential of using aportable sequencing device in the field for tracking faecal contamination.
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