Trichloroethylene

The genotypic results were interpreted using three commonly used rule-base interpretation systems: Agence Nationale de recherches sur le SIDA (ANRS) version 17; Stanford HIVdb, version 5.1.2; and Rega Institute version 8.0.1. The ANRS and Rega both report 3 levels of resistance: susceptible, intermediate, and resistant. For ANRS, we translated the definitions ‘susceptible’, ‘intermediate’, and ‘resistant’ into susceptibility scores of 1, 0.5, and 0, respectively. For the Rega scores, we used the weighted score suggested by Rega, which uses the following changes: NNRTI were scored 0.25 (with the exception of etravirine with a score of 0.5) for intermediate resistance, and ritonavir-boosted PI were scored 0.75 and 1.5 for intermediate resistance and susceptible, respectively. The Stanford algorithm uses 5 levels of resistance. We assigned the following scores to these 5 levels of Stanford: 0, 0.25, 0.50, 0.75, and 1 for respectively the high-level resistance, intermediate resistance, low-level resistance, potential low-level resistance, and susceptible. In a separate analysis we used the unweighted scores for Rega. We assigned the scores 0, 0.5, and 1 to the ‘resistant’, ‘intermediate’, and ‘susceptible’ groups for all drugs, respectively. The three systems did not include a score for ritonavir. We therefore excluded eleven TCEs that used ritonavir as only protease inhibitor, as we could not calculate a GSS of their treatment regimens.
The arithmetic sum of the individual score for the specific drugs provided the total GSS of that treatment. For brevity, we classified the total GSS score in the following categories: 0 to <1, 1 to <2, 2 to <3, 3 to <4, and ≥4. The 0 to <1 group contains viral sequences almost entirely resistant to the drugs in their regimen, and the ≥4 group contains viral sequences susceptible to more than 3 drugs given in their regimen.
To calculate the prevalence of drug resistance we used the mutation list published by the International AIDS Society USA (IAS-USA) [13] (link).

The KB-1 consortium was maintained in batch culture in defined mineral medium
[65 (link)] with trichloroethene (TCE) as electron acceptor and methanol as electron donor. The culture was routinely allowed to dechlorinate TCE completely to ethene prior to a new amendment of acceptor/donor approximately every two weeks. The DonnaII reactor and ANAS semi-batch reactor were maintained as described previously
[19 (link),20 (link)]. See Table
1 for a summary comparison of maintenance conditions, and Table
2 for metagenome sizes and modes of sequencing used.
For the KB-1 metagenome, DNA was extracted from the T3 MP1 KB-1 culture just after completion of a dechlorination cycle using a Cetyl trimethylammonium bromide (CTAB) protocol
[66 ] with volumes scaled up for higher yield as described in the alternate protocol, omitting subsequent cesium chloride gradient centrifugation steps. Clone libraries with 35-kb and 3-kb inserts were created by the JGI using their in-house protocols (
http://www.jgi.doe.gov/sequencing/protocols), and an additional 3 kb short insert library generated for construction of a shotgun metagenome microarray was constructed by Genome Atlantic. A total of 103 MB of metagenome sequence was generated on AB13730xl Sanger sequencers from the three clone libraries. The metagenome was assembled using the JGI’s in-house bacterial assembly pipeline, utilizing lucy
[67 (link)] for vector and quality screening. The KB-1 metagenome sequence and assembly were made publically available by the JGI on May 2^nd, 2009 (
http://genome.jgi-psf.org/aqukb/aqukb.download.ftp.html). Community composition of the three KB-1 DNA samples utilized for sequencing the KB-1 metagenome was determined by Dr. Alison S. Waller using qPCR
[30 ].
The DonnaII metagenome was generated from 454 Titanium libraries according to the JGI’s in-house protocols (
http://www.jgi.doe.gov/sequencing/protocols). Metagenome assembly was conducted using the Newbler program from Roche
[68 (link)]. The sequence data, including an in-house assembly draft for the DonnaII metagenome was made publically available by the JGI on January 31^st, 2012 (IMG-M taxon ID: 2032320001 (
http://img.jgi.doe.gov/cgi-bin/m/main.cgi)).
The ANAS metagenome was composed of a combination of Sanger sequencing and Titanium 454 sequencing as described above. The sequence data, including an in-house assembly draft for the ANAS metagenome was made publically available by the JGI on August 20^th, 2009 (IMG-M taxon ID: 2014730001 (
http://img.jgi.doe.gov/cgi-bin/m/main.cgi)).

GBD risk factors are estimated based on a comparative risk assessment framework which includes six steps. First, the identification of risk-outcome pairs: only those risk-outcomes, which have convincing or plausible evidence according to the World Cancer Research Fund criteria (12 ), will be involved in GBD risk factor estimation. Second, relative risk (RR) as a function of exposure for each risk-outcome pair is estimated. Third, exposure for each risk factor is distributed by age, sex, location, and year. Fourth, the theoretical minimum risk exposure level (TMREL) is demonstrated. Fifth, the population attributable fraction (PAF) and attributable burden are measured. The PAF is modeled by the RR for each risk-outcome pair, exposure levels, and TMRE L (9 (link)). The PAF of a particular risk factor is compounded by genitourinary cancer mortality to engender the mortality attributable to that risk factor. Finally, the PAF and attributable burden for the combination of risk factors are estimated. The methodology of these steps has been comprehensively reviewed in previously published articles (9 (link)). Four [high body-mass index (BMI) for kidney cancer, occupational exposure to trichloroethylene for kidney cancer, high fasting plasma glucose for bladder cancer, and smoking for kidney, bladder and prostate cancer] of the 87 risk factors included in this GBD iteration have a non-zero contribution to the mortality of genitourinary cancers deaths. The percentage contribution of these four risk factors to genitourinary cancers is assessed.