Diverse and Widespread Contamination Evident in the Unmapped Depths of High Throughput Sequencing Data

Abstract

Trace quantities of contaminating DNA are widespread in the laboratory environment, but their presence has received little attention in the context of high throughput sequencing. This issue is highlighted by recent works that have rested controversial claims upon sequencing data that appear to support the presence of unexpected exogenous species. I used reads that preferentially aligned to alternate genomes to infer the distribution of potential contaminant species in a set of independent sequencing experiments. I confirmed that dilute samples are more exposed to contaminating DNA, and, focusing on four single-cell sequencing experiments, found that these contaminants appear to originate from a wide diversity of clades. Although negative control libraries prepared from 'blank' samples recovered the highest-frequency contaminants, low-frequency contaminants, which appeared to make heterogeneous contributions to samples prepared in parallel within a single experiment, were not well controlled for. I used these results to show that, despite heavy replication and plausible controls, contamination can explain all of the observations used to support a recent claim that complete genes pass from food to human blood. Contamination must be considered a potential source of signals of exogenous species in sequencing data, even if these signals are replicated in independent experiments, vary across conditions, or indicate a species which seems a priori unlikely to contaminate. Negative control libraries processed in parallel are essential to control for contaminant DNAs, but their limited ability to recover low-frequency contaminants must be recognized.

Full text

Its licence does not allow us to show the full text. Read it at PubMed Central:

References 43

1. 1.

   Schmidt T, Hummel S, Herrmann B (1995 ) Evidence of contamination in PCR laboratory disposables. Naturwissenschaften 82(9): 423–31.

   Not in our library10.1007/bf01133677

2. 2.

   Leonard JA, Shanks O, Hofreiter M, Kreuz E, Hodges L, et al (2007 ) Animal DNA in PCR reagents plagues ancient DNA research. Journal of Archaeological Science 34(9): 1361–6.

   Not in our library

3. 3.

   Peters RP, Mohammadi T, Vandenbroucke Grauls CM, Danner SA, van Agtmael MA, et al (2004 ) Detection of bacterial DNA in blood samples from febrile patients: underestimated infection or emerging contamination? FEMS Immunol Med Microbiol 42(2): 249–53.

   Not in our library10.1016/j.femsim.2004.05.009

4. 4.

   Ehricht R, Hotzel H, Sachse K, Slickers P (2007 ) Residual DNA in thermostable DNA polymerases - a cause of irritation in diagnostic PCR and microarray assays. Biologicals 35(2): 145–7.

   Not in our library10.1016/j.biologicals.2006.04.001

5. 5.

   Evans GE, Murdoch DR, Anderson TP, Potter HC, George PM, et al (2003 ) Contamination of Qiagen DNA extraction kits with Legionella DNA. J Clin Microbiol 41(7): 3452–3.

   Not in our library10.1128/jcm.41.7.3452-3453.2003

6. 6.

   Hughes MS, Beck LA, Skuce RA (1994 ) Identification and elimination of DNA sequences in Taq DNA polymerase. J Clin Microbiol 32(8): 2007–8.

   Not in our library10.1128/jcm.32.8.2007-2008.1994

7. 7.
   DNA extraction columns contaminated with murine sequences. · 2011

   Not checked
8. 8.

   Persing DH (1991 ) Polymerase chain reaction: trenches to benches. J Clin Microbiol 29(7): 1281–5.

   Not in our library10.1128/jcm.29.7.1281-1285.1991

9. 9.
   An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications · 2010

   No notice
10. 10.

    Gill P, Whitaker J, Flaxman C, Brown N, Buckleton J (2000 ) An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA. Forensic Sci Int 112(1): 17–40.

    Not in our library10.1016/s0379-0738(00)00158-4

11. 11.

    Corless CE, Guiver M, Borrow R, Edwards-Jones V, Kaczmarski EB, et al (2000 ) Contamination and sensitivity issues with a real-time universal 16S rRNA PCR. J Clin Microbiol 38(5): 1747–52.

    Not in our library10.1128/jcm.38.5.1747-1752.2000

12. 12.

    Longo MS, O'Neill MJ, O'Neill RJ (2011 ) Abundant human DNA contamination identified in non-primate genome databases. PLoS One 6(2): e16410.

    Not in our library10.1371/journal.pone.0016410

13. 13.

    Binns M (1993 ) Contamination of DNA database sequence entries with Escherichia coli insertion sequences. Nucleic Acids Res 21(3): 779.

    Not in our library10.1093/nar/21.3.779

14. 14.
    Fast Identification and Removal of Sequence Contamination from Genomic and Metagenomic Datasets · 2011

    No notice
15. 15.

    Astua-Monge G, Lyznik A, Jones V, Mackenzie SA, Vallejos CE (2002 ) Evidence for a prokaryotic insertion-sequence contamination in eukaryotic sequences registered in different databases. Theor Appl Genet 104(1): 48–53.

    Not in our library10.1007/s001220200005

16. 16.

    Percudani R (2013 ) A Microbial Metagenome (Leucobacter sp.) in Caenorhabditis Whole Genome Sequences. Bioinform Biol Insights 7: 55–72.

    Not in our library10.4137/bbi.s11064

17. 17.

    Tosar JP, Rovira C, Naya H, Cayota A (2014 ) Mining of public sequencing databases supports a non-dietary origin for putative foreign miRNAs: underestimated effects of contamination in NGS. RNA 20: 754–7.

    Not in our library10.1261/rna.044263.114

18. 18.

    Parkinson NJ, Maslau S, Ferneyhough B, Zhang G, Gregory L, et al (2012 ) Preparation of high-quality next-generation sequencing libraries from picogram quantities of target DNA. Genome Res 20: 754–7.

    Not in our library10.1101/gr.124016.111

19. 19.
    Ultrafast and memory-efficient alignment of short DNA sequences to the human genome · 2009

    No notice
20. 20.

    International Human Genome Sequencing Consortium (2001 ) Initial sequencing and analysis of the human genome. Nature 409(6822): 860–921.

    Not in our library10.1038/35057062

21. 21.

    Marschner IC (2011 ) glm2: fitting generalized linear models with convergence problems. The R journal 3(2): 12–5.

    Not in our library

22. 22.

    R Core Team (2014) R: A language and environment for statistical computing. Vienna: the R Foundation for Statistical Computing. 3397 p.

    Not in our library

23. 23.

    Navin N, Kendall J, Troge J, Andrews P, Rodgers L, et al (2011 ) Tumour evolution inferred by single-cell sequencing. Nature 472(7341): 90–4.

    Not in our library10.1038/nature09807

24. 24.

    Falconer E, Hills M, Naumann U, Poon SS, Chavez EA, et al (2012 ) DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution. Nat Methods 9(11): 1107–12.

    Not in our library10.1038/nmeth.2206

25. 25.

    Islam S, Kjällquist U, Moliner A, Zajac P, Fan JB, et al (2011 ) Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Res 21(7): 1160–7.

    Not in our library10.1101/gr.110882.110

26. 26.

    Lu S, Zong C, Fan W, Yang M, Li J, et al (2012 ) Probing meiotic recombination and aneuploidy of single sperm cells by whole-genome sequencing. Science 338(6114): 1627–30.

    Not in our library10.1126/science.1229112

27. 27.

    Mouse genome sequencing consortium (2002 ) Initial sequencing and analysis of the mouse genome. Nature 420(6915): 520–62.

    Not in our library10.1038/nature01262

28. 28.

    Spisák S, Solymosi N, Ittzés P, Bodor A, Kondor D, et al (2013 ) Complete genes may pass from food to human blood. PLoS One 8(7): e69805.

    Not in our library10.1371/journal.pone.0069805

29. 29.

    Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al (1997 ) Gapped Blast and psi-Blast: A new generation of protein database search programs. Nucleic Acids Res 25: 3389–402.

    Not in our library10.1093/nar/25.17.3389

30. 30.

    Munch K, Boomsma W, Huelsenbeck JP, Willerslev E, Nielsen R (2008 ) Statistical assignment of DNA sequences using Bayesian phylogenetics. Syst Biol 57(5): 750–7.

    Not in our library10.1080/10635150802422316

31. 31.

    OBITool Development Team. Available: http://www.grenoble.prabi.fr/trac/OBITools. Accessed 2014 August 17.

    Not in our library

32. 32.
    Serendipitous discovery of Wolbachia genomes in multiple Drosophilaspecies · 2005

    No notice
33. 33.

    Kircher M, Sawyer S, Meyer M (2012 ) Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic Acids Rsch 40(1): e3.

    Not in our library10.1093/nar/gkr771

34. 34.

    Combs PA, Eisen MB (2013 ) Sequencing mRNA from cryo-sliced Drosophila embryos to determine genome-wide spatial patterns of gene expression. PLoS One 8(8): e71820.

    Not in our library10.1371/journal.pone.0071820

35. 35.

    Salazar LA, Hirata MH, Cavalli SA, Machado MO, Hirata RD (1998 ) Optimized procedure for DNA isolation from fresh and cryopreserved clotted human blood useful in clinical molecular testing. Clin Chem 44(8): 1748–50.

    Not in our library

36. 36.

    Cherepanova AV, Tamkovich SN, Bryzgunova OE, Vlassov VV, Laktionov PP (2008 ) Deoxyribonuclease activity and circulating DNA concentration in blood plasma of patients with prostate tumors. Ann N Y Acad Sci 1137: 218–21.

    Not in our library10.1196/annals.1448.016

37. 37.

    Cogen AL, Nizet V, Gallo RL (2009 ) Skin microbiota: a source of disease or defence? Br J Dermatology 158(3): 442–55.

    Not in our library10.1111/j.1365-2133.2008.08437.x

38. 38.

    Yim SM, Kim JY, Ko JH, Lee YW, Choe YB, et al (2010 ) Molecular analysis of Malassezia microflora on the skin of the patients with atopic dermatitis. Ann Dermatol 22(1): 41–7.

    Not in our library10.5021/ad.2010.22.1.41

39. 39.

    Gossling J, Slack JM (1974 ) Predominant Gram-Positive Bacteria in Human Feces: Numbers, Variety, and Persistence. Infection and Immunity 9(4): 719–29.

    Not in our library10.1128/iai.9.4.719-729.1974

40. 40.

    Gouba N, Raoult D, Drancourt M (2013 ) Plant and fungal diversity in gut microbiota as revealed by molecular and culture investigations. PLOS ONE 8(3): e59474.

    Not in our library10.1371/journal.pone.0059474

Cited by papers we hold 16

• Is There a Brain Microbiome? · 2021
  Not checked
• Massive gene presence-absence variation shapes an open pan-genome in the Mediterranean mussel · 2020
  No notice
• A Lot on Your Plate? Well-to-Well Contamination as an Additional Confounder in Microbiome Sequence Analyses · 2019
  Not checked
• A systematic sequencing-based approach for microbial contaminant detection and functional inference · 2019
  Not checked
• Issues and current standards of controls in microbiome research · 2019
  No notice
• Controlling for Contaminants in Low-Biomass 16S rRNA Gene Sequencing Experiments · 2019
  No notice
• Access to RNA-sequencing data from 1,173 plant species: The 1000 Plant transcriptomes initiative (1KP) · 2019
  No notice
• Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data · 2018
  No notice
• 16S rRNA Next Generation Sequencing Analysis Shows Bacteria in Alzheimer’s Post-Mortem Brain · 2017
  No notice
• The blood DNA virome in 8,000 humans · 2017
  No notice
• Taxonomer: an interactive metagenomics analysis portal for universal pathogen detection and host mRNA expression profiling · 2016
  No notice
• Characterization of shed medicinal leech mucus reveals a diverse microbiota · 2015
  Not checked
• Composition, taxonomy and functional diversity of the oropharynx microbiome in individuals with schizophrenia and controls · 2015
  No notice
• Microbiota of the indoor environment: a meta-analysis · 2015
  No notice
• Functional Characterization of Bacteria Isolated from Ancient Arctic Soil Exposes Diverse Resistance Mechanisms to Modern Antibiotics · 2015
  No notice
• Large-scale contamination of microbial isolate genomes by Illumina PhiX control · 2015
  No notice

Reads

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.