Open Access
Open Peer Review

This article has Open Peer Review reports available.

How does Open Peer Review work?

Genomic resources and draft assemblies of the human and porcine varieties of scabies mites, Sarcoptes scabiei var. hominis and var. suis

  • Ehtesham Mofiz1, 2,
  • Deborah C. Holt3,
  • Torsten Seemann4,
  • Bart J. Currie3,
  • Katja Fischer5 and
  • Anthony T. Papenfuss1, 2, 6, 7Email author
Contributed equally
GigaScience20165:23

DOI: 10.1186/s13742-016-0129-2

Received: 18 January 2016

Accepted: 11 May 2016

Published: 2 June 2016

Abstract

Background

The scabies mite, Sarcoptes scabiei, is a parasitic arachnid and cause of the infectious skin disease scabies in humans and mange in other animal species. Scabies infections are a major health problem, particularly in remote Indigenous communities in Australia, where secondary group A streptococcal and Staphylococcus aureus infections of scabies sores are thought to drive the high rate of rheumatic heart disease and chronic kidney disease.

Results

We sequenced the genome of two samples of Sarcoptes scabiei var. hominis obtained from unrelated patients with crusted scabies located in different parts of northern Australia using the Illumina HiSeq. We also sequenced samples of Sarcoptes scabiei var. suis from a pig model. Because of the small size of the scabies mite, these data are derived from pools of thousands of mites and are metagenomic, including host and microbiome DNA. We performed cleaning and de novo assembly and present Sarcoptes scabiei var. hominis and var. suis draft reference genomes. We have constructed a preliminary annotation of this reference comprising 13,226 putative coding sequences based on sequence similarity to known proteins.

Conclusions

We have developed extensive genomic resources for the scabies mite, including reference genomes and a preliminary annotation.

Keywords

Scabies mite Sarcoptes scabiei var. hominis Sarcoptes scabiei var. suis Indigenous Australian health

Data description

The scabies mite, Sarcoptes scabiei, is an ectoparasitic acari, which causes rashes and extreme itching - known as scabies in humans. Different varieties of the scabies mite also cause mange in other species of mammals including domestic animals, livestock and wildlife. Scabies is known to cause significant morbidity in some populations, in particular Indigenous communities in Australia. We present extensive genomic sequencing data from human (Sarcoptes scabiei var. hominis) and pig (Sarcoptes scabiei var. suis) varieties of scabies mites, including Illumina whole genome sequencing data from two independent samples of adult scabies mites collected at different times from human patients from different regions of northern Australia, and from four samples of scabies mites from a pig model collected at different times and washed using different protocols to reduce bacterial contamination from host skin and mite gut. We created draft genome assemblies for var. hominis and var. suis from these resources.

Samples and sequencing

Scabies mites (var. hominis) were individually picked from skin scrapings collected 14 months apart from two unrelated patients from two different regions of northern Australia with severe crusted scabies (Patients A and B). Over 1000 mites were collected in each sample. Two pig mange mite (var. suis) samples were collected from an inbred population of mites from a pig model [1]. The first sample consisted of >1000 mites from adult, nymph, larva and egg life stages (Pig Unwashed). The second sample, also containing all life stages, was split into three subsamples that were washed - to reduce the amount of bacteria present on the surface of the mites owing to the wound micro-environment - using three different protocols (Pig Washed 1, 2 and 3): (i) 15 min wash at room temperature in 4 % paraformaldehyde in water [2]; (ii) 1 h incubation at 37 °C in 150 mM NaCl, 10 mM EDTA pH8.0, 0.6 % SDS and 0.125 μg/μl lysozyme [3]; (iii) 1 h incubation at 37 °C in 1 % bleach (sodium hypochlorite) in water. In all protocols, mites were subsequently rinsed twice in water. Between wash steps, mites were centrifuged at 10,000 rpm for 2 min.

Whole mites were crushed and DNA was extracted from each sample using a QIAGEN Blood and Cell Culture DNA Kit and a modified procedure adapted from the manufacturer’s protocol. Washed mites were submerged in 1 ml of ice-cold lysis buffer (20 mM EDTA, 100 mM NaCl, 1 % TritonX-100, 500 mM guanidine-HCl, 10 mM Tris pH7.9) and homogenized with stainless steel beads of 2.8 mm diameter at 6800 rpm, three cycles, 30 s per cycle, and 30 s between cycles. The suspension of lysed mites was supplemented with DNase-free RNase A to 0.2 mg/ml and with proteinase K to 0.8 mg/ml and incubated at 50 °C for 1.5 h. After centrifugation at 4000 × g for 10 min to pellet insoluble debris, the genomic DNA was isolated on the QIAGEN genomic tip as per the manufacturer’s protocol. Six DNA libraries were constructed and 100-base pair (bp) paired-end reads were generated using an Illumina HiSeq 2500 (see Table 1 for details).
Table 1

Details of sequencing libraries

Sample type

Label

Washing protocol

Number of read pairs

Clinical isolate

Patient A

-

53,699,468

Clinical isolate

Patient B

-

45,851,518

Lab model

Pig Unwashed

-

59,011,146

Lab model

Pig Washed 1

Paraformaldehyde

62,090,067

Lab model

Pig Washed 2

Lysozyme

56,485,415

Lab model

Pig Washed 3

Bleach

55,580,620

Genome assembly

Read qualities were assessed using FASTQC [4], and reads were adapter- and quality-trimmed (Q ≥ 20) using Trim Galore! (v3.0.1) [5].

Preliminary de novo assemblies of the adapter- and quality-trimmed reads of the Patient A, Patient B and Pig Unwashed samples were performed by using Velvet (v1.2.08) [6]. For the Patient B library, k-mer values of 61, 63, 65, 67, 69, 71, 73, 75, 79, 85, 89 and 95 were used. For the Patient A and three Pig Unwashed libraries, k-mer values of 69, 75, 77, 79, 81, 83, 85, 89 and 95 were used. The best assemblies (assessed using the scaffold N50) were obtained with a k-mer of k = 77 (Patient A, N50 = 27.4 kb), k = 63 (Patient B, N50 = 36.0 kb) and k = 81 (Pig Unwashed, N50 = 7.5 kb) (see Additional file 1 for details). Platanus (version 1.2.1) [7] was also used to perform a preliminary assembly of all six libraries, producing assemblies with better scaffold N50 values (GigaScience repository [8] for var. suis).

Since the scabies mite is a tiny, obligate parasite, it is difficult to avoid contamination from the host and from host skin and mite gut microbiomes. In addition, it was necessary to sequence thousands of intact mites, which incorporated the mite gut. Reads from the host genome were removed in silico from each sample using Bowtie 2 (version 2.2.5) [9]. Human hg19 and pig susScr3 reference genomes from the University of California, Santa Cruz, were used to build Bowtie 2 reference indices for alignment. For each sample, adapter- and quality-trimmed reads were aligned to the host reference genome using Bowtie 2 (using mode ‘--end-to-end’ and parameter ‘--very-fast’). The proportion of reads aligning to host reference genomes varied from 11 to 56 % (Table 2). Non-host reads were extracted from the alignment SAM files using the SAMtools [10] ‘view’ command with flag ‘-f 12’ (read unmapped, mate unmapped).
Table 2

Summary statistics for host-filtered Platanus assemblies

 

Patient A

Patient B

Pig unwashed

Pig washed 1

Pig washed 2

Pig washed 3

Pig washed pooled

Host filtering using Bowtie 2 alignment

 Host-aligned read percentage

55.68 %

22.51 %

14.20 %

10.99 %

43.98 %

11.07 %

N/A

Scaffolds

 Scaffold N50

29,787

45,917

6352

6835

22,475

36,156

4883

 Largest scaffold

509386

794311

88,812

681,477

423,133

809,115

299,570

 Total assembled bases

68,937,519

61,661,613

69,459,333

68,875,212

61,832,214

56,344,534

75,837,484

 No of scaffolds

99,178

66,591

47,952

149,238

83,245

26,086

212,580

Scaffolds (≥500 bp)

 Major scaffold N50

43,122

62,417

7574

17,034

30,929

40,825

-

 Largest scaffold

509,386

794,311

88,812

681,477

423,133

809,115

-

 Total assembled bases

56,795,385

53,697,990

62,853,857

47,516,449

52,301,800

53,472,496

-

 No of scaffolds

4276

3157

17236

7586

5102

4269

-

Each host-filtered library was then assembled using Platanus (version 1.2.1, default settings), because this method performed better in the preliminary assembly of unfiltered reads. This produced assemblies with scaffold N50s ranging from 6 kb (Pig Unwashed) to 46 kb (Patient B) and major N50s up to 62 kb (see Table 2 for details). A pooled assembly of the three host-filtered washed pig samples (Pig Washed 1, 2 and 3) was also performed, producing an N50 of 4.8 kb.

The Platanus assemblies of Patient B and Pig Washed 3 had the largest major N50s (62.4 kb and 40.8 kb respectively) and were selected as the var. hominis and var. suis draft reference genomes (Table 3).
Table 3

Summary statistics for Sarcoptes scabiei draft reference genomes

Genome

Assembly size (bp)

No of scaffolds

Major scaffold N50 (bp)

Largest scaffold (bp)

No of gene features annotated

Sarcoptes scabiei var. hominis (Patient B)

53,667,537

3138

63,351

794,311

13,226

Sarcoptes scabiei var. suis (Pig Washed 3)

53,470,956

4268

40,825

809,115

-

These two draft assemblies were then filtered for bacterial scaffolds by aligning scaffolds to the National Center for Biotechnology Information (NCBI) Microbial RefSeq database v72 [11] using BLASTN (version 2.2.30+; E-value cutoff 10−20; max_target_seqs = 1) [12]. The best hits in which >80 % of the scaffold length aligned to bacterial sequences were filtered out, removing 19 scaffolds from Patient B and one scaffold from Pig Washed 3. A similar search on the assemblies prior to filtering small contigs showed that most of the bacterial contigs in the assemblies were shorter than 500 bp.

To estimate the proportion of bacterial DNA contaminating the samples, microbial classification was performed on unfiltered reads from each sample using Kraken [13]. Kraken was run with default settings using the standard bacterial, archaeal and viral database (downloaded on 3 November 2014). The samples were found to have contaminant proportions of between 4 and 9 % (Additional file 2).

After removal of bacterial scaffolds, we obtained the final Sarcoptes scabiei var. hominis and var. suis draft genome assemblies, which had final major scaffold N50 values of 63.3 kb (Patient B) and 40.8 kb (Pig Washed 3). The genome sizes of the assemblies were 53.7 Mb in 3138 scaffolds (Patient B) and 53.5 Mb in 4268 scaffolds (Pig Washed 3) (Table 3). Protocols presented here are also available in protocols.io [14].

Estimation of genome completeness

To estimate the completeness of the assemblies, the Core Eukaryotic Genes Mapping Approach (CEGMA) [15] and Benchmarking Universal Single-Copy Orthologs (BUSCO) [16] strategies were applied to the var. hominis and suis draft genome assemblies. CEGMA (v2.5) was run with default settings on both assemblies to estimate genome completeness based on 248 ultra-conserved core eukaryotic genes (CEGs) found in nearly all eukaryotes. For both assemblies, CEGMA estimated 98.79 % completeness based on complete matches and 99.19 % completeness based on partial matches. BUSCO (v1.1b) was run in default settings using single-copy ortholog gene set databases for eukaryote taxonomic group. Seventy-five percent (75 %) of genes from the gene set of eukaryotes were predicted in both the draft genomes (66 % complete and 8.8 % fragmented genes in var. hominis and 67 % complete and 7.9 % fragmented in var. suis).

Preliminary genome annotation

A preliminary annotation of the var. hominis draft genome (Patient B) assembly was constructed by aligning UniProtKB/Swiss-Prot proteins (release 2015_07) [17] with the assembly using TBLASTN (version 2.2.30+; E-value cutoff 10−6) [12]. Multiple annotations intersecting scaffold positions on the same strand were merged into a single annotation using the BEDTools (v2.25.0) [18] ‘merge’ sub-command in strand-specific mode. After the merging step, a total of 13,226 gene features were annotated.

Comparison with other scabies genomics resources

The mitochondrial genome reference sequence for Sarcoptes scabiei var. hominis and var. suis have been published [19] and used to investigate within-patient diversity of infestations. A draft genome assembly of Sarcoptes scabiei var. canis is also available [20]. The scaffold N50 of this genome was 11.6 kb with a largest scaffold of 358.8 kb; the total assembly size was 56.2 Mb with a total of 18,600 scaffolds. In comparison, the var. hominis (Patient B) draft assembly had a scaffold N50 of 63.3 kb with a largest scaffold of 794.3 kb; the total assembly size was 53.6 Mb with a total of 3138 scaffolds. The annotation of the var. canis genome consisted of 10,644 predicted protein-coding genes, and the preliminary annotation of the var. hominis genome consists of 13,226 gene similarity features. The var. canis assembly had an estimated completeness of 93.55 % using CEGMA, while both var. hominis and var. suis draft genome assemblies had 99.19 and 98.79 % completeness based on partial and complete matches respectively.

Availability of supporting data

Supporting data is available in the GigaScience repository [8] and raw data in NCBI (BioProject accession: PRJEB12428). Genome assembly protocols presented here are also archived in protocols.io [14].

Ethics approval and consent to participate

The collection of human patient samples was approved by the Human Research Ethics Committee of the Northern Territory Department of Health and Menzies School of Health Research (approval 13–2027), and informed consent was obtained from each participant. Animal care and handling procedures used in this study followed the Animal Care and Protection Act, in compliance with the Australian code of practice for the care and use of animals for scientific purposes, outlined by the Australian National Health and Medical Research Council (NHMRC). The study was approved by the Queensland Animal Science Precinct (QASP) and the QIMR Berghofer MRI Animal Ethics Committees (DEEDIAEC SA2012/02/381, QIMR A0306-621 M).

Notes

Abbreviations

bp: 

base pairs

BUSCO: 

Benchmarking Universal Single-Copy Orthologs

CEGMA: 

core eukaryotic genes mapping approach

CEGs: 

core eukaryotic genes

NCBI: 

National Center for Biotechnology Information

Declarations

Acknowledgements

This research was supported by The Scobie and Claire Mackinnon Trust, the Lettisier Foundation, the Evans Family Foundation and the Australian NHMRC Program Grants (1054618 and 496600). KF and MB were supported by Australian Research Council Future Fellowships. The research benefitted from the support of Victorian State Government Operational Infrastructure Support and Australian Government NHMRC Independent Research Institute Infrastructure Support. We acknowledge Mr Andrew Kelly and Mrs Beverly Hutchinson for the animal management at the QASP.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research
(2)
Department of Medical Biology, University of Melbourne
(3)
Menzies School of Health Research, Charles Darwin University
(4)
Victorian Life Sciences Computation Initiative, University of Melbourne
(5)
QIMR Berghofer Medical Research Institute
(6)
Sir Peter MacCallum Department of Oncology, University of Melbourne
(7)
Peter MacCallum Cancer Centre

References

  1. Mounsey K, Ho MF, Kelly A, Willis C, Pasay C, Kemp DJ, et al. A tractable experimental model for study of human and animal scabies. PLoS Negl Trop Dis. 2010;4(7), e756.View ArticlePubMedPubMed CentralGoogle Scholar
  2. Moro CV, Chauve C, Zenner L. Experimental infection of Salmonella Enteritidis by the poultry red mite, Dermanyssus gallinae. Vet Parasitol. 2007;146(3):329–36.View ArticleGoogle Scholar
  3. Reed D, Hafner M. Phylogenetic analysis of bacterial communities associated with ectoparasitic chewing lice of pocket gophers: a culture-independent approach. Microb Ecol. 2002;44(1):78–93.View ArticlePubMedGoogle Scholar
  4. Harvey MS. The neglected cousins: what do we know about the smaller arachnid orders? J Arachnol. 2002;30(2):357–72.View ArticleGoogle Scholar
  5. Dermauw W, Van Leeuwen T, Vanholme B, Tirry L. The complete mitochondrial genome of the house dust mite Dermatophagoides pteronyssinus (Trouessart): a novel gene arrangement among arthropods. BMC Genomics. 2009;10:107.View ArticlePubMedPubMed CentralGoogle Scholar
  6. Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008;18(5):821–9.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Kajitani R, Toshimoto K, Noguchi H, Toyoda A, Ogura Y, Okuno M, et al. Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads. Genome Res. 2014;24(8):1384–95.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Mofiz E, Holt D, Seemann T, Currie BJ, Fischer K, Papenfuss AT. The data for: Genomic resources and draft reference assemblies of the human and porcine scabies mites, Sarcoptes scabiei var. hominis and var. suis GigaScience Database. http://dx.doi.org/10.5524/100198; 2016
  9. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357–9.View ArticlePubMedPubMed CentralGoogle Scholar
  10. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25(16):2078–9.View ArticlePubMedPubMed CentralGoogle Scholar
  11. Tatusova T, Ciufo S, Fedorov B, O’Neill K, Tolstoy I. RefSeq microbial genomes database: new representation and annotation strategy. Nucleic Acids Res. 2014;42(D1):D553–D559.
  12. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009;10:421.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Wood D, Salzberg S. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol. 2014;15(3):R46.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Mofiz E, Holt D, Seemann T, Currie BJ, Fischer K, Papenfuss AT. Draft genome assembly using parasitic mite population NGS DNA sample from mites extracted from host wound environment. protocols.io. http://dx.doi.org/10.17504/protocols.io.exwbfpe; 2016.
  15. Parra G, Bradnam K, Korf I. CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics. 2007;23(9):1061–7.View ArticlePubMedGoogle Scholar
  16. Simão FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015;31(19):3210–2.View ArticlePubMedGoogle Scholar
  17. UniProt Consortium. UniProt: a hub for protein information. Nucleic Acids Res. 2015;43(D1):D204–D212.
  18. Quinlan AR. BEDTools: the Swiss‐army tool for genome feature analysis. Curr Protoc Bioinformatics. 2014;47:11.12.1–34.
  19. Mofiz E, Seemann T, Bahlo M, Holt D, Currie BJ, Fischer K, et al. Mitochondrial genome sequence of the scabies mite provides insight into the genetic diversity of individual scabies infections. PLoS Negl Trop Dis. 2016;10(2), e0004384.View ArticlePubMedPubMed CentralGoogle Scholar
  20. Rider Jr SD, Morgan MS, Arlian LG. Draft genome of the scabies mite. Parasites Vectors. 2015;8(1):1–14.View ArticleGoogle Scholar

Copyright

© Mofiz et al. 2016