Here, we present a comprehensive transcriptomics data set of Brugia malayi, its Wolbachia endosymbiont wBm, and its vector host. This study samples from 16 stages across the entire B. malayi life cycle, including stage 1 through 4 larvae, adult males and females, embryos, immature microfilariae, and mature microfilariae.
ABSTRACT
Here, we present a comprehensive transcriptomics data set of Brugia malayi, its Wolbachia endosymbiont wBm, and its vector host. This study samples from 16 stages across the entire B. malayi life cycle, including stage 1 through 4 larvae, adult males and females, embryos, immature microfilariae, and mature microfilariae.
ANNOUNCEMENT
Brugia malayi is the laboratory model for lymphatic filariasis, one of the most prevalent vector-borne parasitic diseases (1) with more than 856 million individuals at risk in regions of endemicity (2). Here, we present transcriptomic data for B. malayi, its Wolbachia endosymbiont wBm, and the vector host across the entire B. malayi life cycle.
B. malayi were isolated using National Institute of Allergy and Infection Diseases/National Institutes of Health (NIAID/NIH) Filariasis Research Reagent Resource Center (FR3) protocols (www.filariasiscenter.org); all animal care and use protocols were approved by the University of Wisconsin Oshkosh Institutional Animal Care and Use Committee (UWO IACUC). All samples were flash frozen in liquid nitrogen and stored at −80°C before RNA isolation. Aedes aegypti black-eyed, Liverpool-strain mosquitoes were fed on FR3-strain microfilaremic cat blood. Infective third-stage B. malayi larvae (L3) were isolated from whole mosquitoes in bulk and used for infections. Thoraces of infected mosquitoes were collected 18 h postinfection (hpi), 4 days postinfection (dpi), and 8 dpi (Table 1). Male gerbils (Meriones unguiculatus) 3 months old or older from Charles River Laboratories were infected with bulk-purified L3s injected into the peritoneal cavity with sample collection at the required times by terminal worm recovery (Table 1). Eggs and embryos were obtained by cutting adult females and removing uterine tissue using forceps (3).
TABLE 1.
RNA-Seq samples and SRA accession numbers across the B. malayi life cycle
| Sample | BioSample accession no. |
Enrichment method | SRA accession no. | No. of total sequenced reads |
|---|---|---|---|---|
| Jird, 1 dpi | SAMN04313629 | Poly(A) enrichment | SRX2409229 | 11,702,630 |
| SAMN04313629 | Poly(A) enrichment | SRX2409230 | 39,429,554 | |
| SAMN04313633 | Poly(A) enrichment | SRX2415911 | 10,512,230 | |
| SAMN04313633 | Poly(A) enrichment | SRX2415910 | 45,513,518 | |
| SAMN04314682 | RiboZero treated, poly(A) depletion | SRX2415979 | 17,479,084 | |
| SAMN04314682 | RiboZero treated, poly(A) depletion | SRX2415978 | 30,356,424 | |
| SAMN04314686 | RiboZero treated, poly(A) depletion | SRX2416090 | 14,570,410 | |
| SAMN04314686 | RiboZero treated, poly(A) depletion | SRX2416089 | 60,046,756 | |
| Jird, 2 dpi | SAMN04313635 | Poly(A) enrichment | SRX2414981 | 12,861,524 |
| SAMN04313635 | Poly(A) enrichment | SRX2414982 | 42,369,518 | |
| SAMN04313631 | Poly(A) enrichment | SRX2415908 | 13,638,502 | |
| SAMN04313631 | Poly(A) enrichment | SRX2415909 | 47,714,364 | |
| SAMN04314688 | RiboZero treated, poly(A) depletion | SRX2416010 | 11,695,030 | |
| SAMN04314688 | RiboZero treated, poly(A) depletion | SRX2416009 | 44,550,904 | |
| SAMN04314684 | RiboZero treated, poly(A) depletion | SRX2416087 | 2,502,950 | |
| SAMN04314684 | RiboZero treated, poly(A) depletion | SRX2416088 | 53,819,452 | |
| Jird, 3 dpi | SAMN04313619 | Poly(A) enrichment | SRX1539755 | 85,614,594 |
| SAMN04313620 | Poly(A) enrichment | SRX1539758 | 97,373,488 | |
| SAMN04314672 | RiboZero treated, poly(A) depletion | SRX1539741 | 31,671,524 | |
| SAMN04314673 | RiboZero treated, poly(A) depletion | SRX1539742 | 36,061,876 | |
| Jird, 4 dpi | SAMN04313621 | Poly(A) enrichment | SRX1539799 | 87,831,322 |
| SAMN04313622 | Poly(A) enrichment | SRX1539813 | 77,919,818 | |
| SAMN04314674 | RiboZero treated, poly(A) depletion | SRX1539745 | 18,001,022 | |
| SAMN04314674 | RiboZero treated, poly(A) depletion | SRX1539744 | 18,059,474 | |
| SAMN04314675 | RiboZero treated, poly(A) depletion | SRX1539747 | 26,818,742 | |
| Jird, 8 dpi | SAMN04313623 | Poly(A) enrichment | SRX1539817 | 84,250,470 |
| SAMN04313624 | Poly(A) enrichment | SRX1539819 | 84,231,566 | |
| SAMN04314676 | RiboZero treated, poly(A) depletion | SRX1539748 | 41,578,604 | |
| SAMN04314677 | RiboZero treated, poly(A) depletion | SRX1539750 | 33,505,904 | |
| Jird, 20 dpi, immature male | SAMN04313625 | Poly(A) enrichment | SRX1539862 | 86,565,686 |
| SAMN04313626 | Poly(A) enrichment | SRX1539865 | 93,010,092 | |
| SAMN04314678 | RiboZero treated, poly(A) depletion | SRX1539752 | 58,212,484 | |
| SAMN04314679 | RiboZero treated, poly(A) depletion | SRX1539754 | 53,899,664 | |
| Jird, 24 dpi, immature female | SAMN04313627 | Poly(A) enrichment | SRX1539869 | 80,399,030 |
| SAMN04313628 | Poly(A) enrichment | SRX1539871 | 94,819,216 | |
| SAMN04314680 | RiboZero treated, poly(A) depletion | SRX1539757 | 73,230,576 | |
| SAMN04314680 | Total RNA, wBm Agilent SureSelect | SRX2508257 | 124,210,676 | |
| SAMN04314681 | RiboZero treated, poly(A) depletion | SRX1539735 | 77,618,810 | |
| Jird, adult male | SAMN04313616 | Poly(A) enrichment | SRX1539740 | 110,125,724 |
| SAMN04313615 | Poly(A) enrichment | SRX1539737 | 95,537,492 | |
| SAMN04314669 | RiboZero treated, poly(A) depletion | SRX1539735 | 77,618,810 | |
| SAMN04314668 | RiboZero treated, poly(A) depletion | SRX1539732 | 43,412,692 | |
| Jird, adult female | SAMN04313613 | Poly(A) enrichment | SRX1539730 | 85,725,576 |
| SAMN04313614 | Poly(A) enrichment | SRX1539734 | 90,360,386 | |
| SAMN04313611 | Poly(A) enrichment | SRX1539085 | 87,158,676 | |
| SAMN04313612 | Poly(A) enrichment | SRX1539707 | 89,298,942 | |
| SAMN04314666 | RiboZero treated, poly(A) depletion | SRX1539729 | 28,901,790 | |
| SAMN04314667 | RiboZero treated, poly(A) depletion | SRX1539731 | 31,173,032 | |
| SAMN04314667 | Total RNA, wBm Agilent SureSelect | SRX2508255 | 147,440,774 | |
| SAMN04314664 | RiboZero treated, poly(A) depletion | SRX1539589 | 52,924,416 | |
| SAMN04314664 | Total RNA, wBm Agilent SureSelect | SRX2508256 | 124,173,684 | |
| SAMN04314665 | RiboZero treated, poly(A) depletion | SRX1539727 | 54,369,010 | |
| Jird, embryo | SAMN04313617 | Poly(A) enrichment | SRX1539746 | 104,139,194 |
| SAMN04313618 | Poly(A) enrichment | SRX1539751 | 103,938,004 | |
| SAMN04314670 | RiboZero treated, poly(A) depletion | SRX1539736 | 38,620,328 | |
| SAMN04314671 | RiboZero treated, poly(A) depletion | SRX1539739 | 43,744,776 | |
| Jird, immature microfilariae | SAMN04313630 | Poly(A) enrichment | SRX2415043 | 12,583,442 |
| SAMN04313630 | Poly(A) enrichment | SRX2415044 | 35,963,192 | |
| SAMN04313632 | Poly(A) enrichment | SRX1539873 | 54,824,638 | |
| SAMN04314683 | RiboZero treated, poly(A) depletion | SRX2416083 | 17,304,704 | |
| SAMN04314683 | RiboZero treated, poly(A) depletion | SRX2416084 | 32,063,516 | |
| SAMN04314685 | RiboZero treated, poly(A) depletion | SRX1539795 | 42,838,356 | |
| Jird, mature microfilariae | SAMN04313636 | Poly(A) enrichment | SRX2416293 | 10,364,900 |
| SAMN04313636 | Poly(A) enrichment | SRX2416292 | 30,366,668 | |
| SAMN04313636 | Poly(A) enrichment | SRX2416292 | 40,464,760 | |
| SAMN04313634 | Poly(A) enrichment | SRX1539875 | 20,074,600 | |
| SAMN04313634 | Poly(A) enrichment | SRX1539874 | 9,974,198 | |
| SAMN04314689 | RiboZero treated, poly(A) depletion | SRX2416086 | 17,610,260 | |
| SAMN04314689 | RiboZero treated, poly(A) depletion | SRX2416085 | 31,206,204 | |
| SAMN04314687 | RiboZero treated, poly(A) depletion | SRX1539797 | 18,534,882 | |
| SAMN04314687 | RiboZero treated, poly(A) depletion | SRX1539798 | 8,452,542 | |
| Vector, 18 hpi | SAMN04313637 | Poly(A) enrichment | SRX1539876 | 103,949,620 |
| SAMN04313637 | Poly(A) enriched, B. malayi Agilent SureSelect |
SRX2505171 | 31,646,034 | |
| SAMN04313637 | Poly(A) enriched, B. malayi Agilent SureSelect |
SRX2505171 | 59,222,150 | |
| SAMN04313637 | Total RNA, B. malayi Agilent SureSelect |
SRX2505170 | 149,907,628 | |
| SAMN04313638 | Poly(A) enrichment | SRX1539886 | 100,358,314 | |
| SAMN04313638 | Poly(A) enriched, B. malayi Agilent SureSelect |
SRX2505769 | 34,175,968 | |
| SAMN04313638 | Poly(A) enriched, B. malayi Agilent SureSelect |
SRX2505769 | 63,720,320 | |
| SAMN04314690 | RiboZero treated, poly(A) depletion | SRX1539809 | 30,623,652 | |
| SAMN04314690 | Total RNA, wBm Agilent SureSelect | SRX2508248 | 76,297,602 | |
| SAMN04314691 | RiboZero treated, poly(A) depletion | SRX1539810 | 30,676,768 | |
| SAMN04314691 | Total RNA, wBm Agilent SureSelect | SRX2508249 | 78,959,932 | |
| Vector, 4 dpi | SAMN04313639 | Poly(A) enrichment | SRX1539947 | 117,570,910 |
| SAMN04313639 | Poly(A) enriched, B. malayi Agilent SureSelect |
SRX2505770 | 57,037,174 | |
| SAMN04313639 | Poly(A) enriched, B. malayi Agilent SureSelect |
SRX2505770 | 106,815,788 | |
| SAMN04313640 | Poly(A) enrichment | SRX1539949 | 113,590,496 | |
| SAMN04313640 | Poly(A) enriched, B. malayi Agilent SureSelect |
SRX2505771 | 48,173,212 | |
| SAMN04313640 | Poly(A) enriched, B. malayi Agilent SureSelect |
SRX2505771 | 90,429,442 | |
| SAMN04314692 | RiboZero treated, poly(A) depletion | SRX1539811 | 38,453,926 | |
| SAMN04314692 | Total RNA, wBm Agilent SureSelect | SRX2508250 | 64,398,742 | |
| SAMN04314693 | RiboZero treated, poly(A) depletion | SRX1539814 | 30,669,394 | |
| SAMN04314693 | Total RNA, wBm Agilent SureSelect | SRX2508252 | 30,161,844 | |
| SAMN04314693 | Total RNA, wBm Agilent SureSelect | SRX2508251 | 21,071,958 | |
| Vector, 8 dpi | SAMN04313641 | Poly(A) enrichment | SRX1539952 | 131,311,710 |
| SAMN04313641 | Poly(A) enriched, B. malayi Agilent SureSelect | SRX2505955 | 87,079,372 | |
| SAMN04313641 | Poly(A) enriched, B. malayi Agilent SureSelect | SRX2505955 | 164,545,760 | |
| SAMN04313641 | Total RNA, B. malayi Agilent SureSelect | SRX2505954 | 255,771,318 | |
| SAMN04313642 | Poly(A) enrichment | SRX1539954 | 146,982,974 | |
| SAMN04313642 | Poly(A) enriched, B. malayi Agilent SureSelect | SRX2505953 | 108,705,568 | |
| SAMN04313642 | Poly(A) enriched, B. malayi Agilent SureSelect | SRX2505953 | 204,486,026 | |
| SAMN04314694 | RiboZero treated, poly(A) depletion | SRX1539815 | 28,267,988 | |
| SAMN04314694 | Total RNA, wBm Agilent SureSelect | SRX2508253 | 74,052,092 | |
| SAMN04314695 | RiboZero treated, poly(A) depletion | SRX1539816 | 41,159,150 | |
| SAMN04314695 | Total RNA, wBm Agilent SureSelect | SRX2508254 | 70,535,594 | |
| Vector, infective L3 | SAMN10039708 | Poly(A) enriched, B. malayi Agilent SureSelect | SRX4676609 | 171,398,760 |
| SAMN10039709 | Poly(A) enriched, B. malayi Agilent SureSelect | SRX4676610 | 171,924,470 |
For RNA isolations, a 3:1 volume of TRIzol was added to mammalian-stage samples, and 1 ml of TRIzol was added per 50 to 100 mg mosquito tissue. We added β-mercaptoethanol (1%), and the tissues were homogenized using a bead beater and a TissueLyser at 50 Hz for 5 min and then centrifuged at 12,000 × g in a fresh tube for 10 min at 4°C. After incubation at room temperature for 5 min, 0.2 volumes of chloroform were added. The samples were shaken by hand for 15 s, incubated at room temperature for 3 min, loaded into a prespun Phase Lock Gel heavy tube, and centrifuged at 12,000 × g for 5 min at 4°C. The upper phase was extracted, 1 volume of 100% ethanol was added, and the sample was purified on a PureLink RNA minicolumn following the manufacturer’s instructions. Purified RNA was run on a bioanalyzer. Microfilariae RNA routinely gives atypical profiles and RNA integrity numbers (RINs). Thus, after test sequencing and analysis, all samples were sequenced regardless of the RIN.
Whole-transcriptome libraries for all samples were constructed using the NEBNext Ultra directional RNA library prep kit. For eukaryotic mRNA, the NEBNext poly(A) mRNA magnetic isolation module was used. For prokaryotic mRNA, rRNA and poly(A) reductions were performed as previously described (4, 5). SPRIselect reagent was used for cDNA purification and size selection, a 7-nucleotide index was added by PCR amplification, and 100-bp paired-end reads were generated with an Illumina HiSeq 2500 instrument. B. malayi and wBm Agilent SureSelect custom bait libraries were designed and used, when necessary, as previously described (6), on poly(A)-selected and total RNA libraries, respectively.
These transcriptomics data form a rich data set that will be of immense value to the filarial nematode and Wolbachia research communities for analyzing gene expression as well as structural annotation of nematode and endosymbiont transcripts.
Data availability.
The data sets supporting the results of this article are available in the Sequence Read Archive (SRA) repository (Table 1). The A. aegypti and B. malayi sequencing reads are available under accession number SRP068692, and the wBm sequencing reads are available under accession number SRP068711.
ACKNOWLEDGMENT
This project was funded by federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under grant number U19 AI110820.
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Associated Data
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Data Availability Statement
The data sets supporting the results of this article are available in the Sequence Read Archive (SRA) repository (Table 1). The A. aegypti and B. malayi sequencing reads are available under accession number SRP068692, and the wBm sequencing reads are available under accession number SRP068711.