Papers of Note from In Sequence, Feb 2009 (4)

Massive parallel bisulfite sequencing of CG-rich DNA fragments reveals that methylation of many X-chromosomal CpG islands in female blood DNA is incomplete.
Michael Zeschnigk, Marcel Martin, Gisela Betzl, Andreas Kalbe, Caroline Sirsch, Karin Buiting, Stephanie Gross, Epameinondas Fritzilas, Bruno Frey, Sven Rahmann, Bernhard Horsthemke.
Hum Mol Genet., Advance Access | doi:10.1093/hmg/ddp054 | PMID:19223391

Methylation of CpG islands (CGIs) plays an important role in gene silencing. For genome-wide methylation analysis of CGIs in female white blood cells and in sperm, we used four restriction enzymes and a size selection step to prepare DNA libraries enriched with CGIs. The DNA libraries were treated with sodium bisulfite and subjected to a modified 454/Roche Genome Sequencer protocol. We obtained 163,034 and 129,620 reads from blood and sperm, respectively, with an average read length of 133 bp. Bioinformatic analysis revealed that 12,358 (7.6%) blood library reads and 10,216 (7.9%) sperm library reads map to 6,167 and 5,796 different CGIs, respectively. In blood and sperm DNA we identified 824 (13.7%) and 482 (8.5%) fully methylated autosomal CGIs, respectively. Differential methylation, which is characterized by the presence of methylated and unmethylated reads of the same CGI, was observed in 53 and 52 autosomal CGIs in blood and sperm DNA, respectively. Remarkably, methylation of X-chromosomal CGIs in female blood cells was most often incomplete (25-75%). Such incomplete methylation was mainly found on the X-chromosome, suggesting that it is linked to X-chromosome inactivation.

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Experimental discovery of sRNAs in Vibrio cholerae by direct cloning, 5S/tRNA depletion and parallel sequencing.
Jane M. Liu, Jonathan Livny, Michael S. Lawrence, Marc D. Kimball, Matthew K. Waldor, Andrew Camilli.
Nucleic Acids Research, Advance Access | doi:10.1093/nar/gkp080 | PMID:19223322

Direct cloning and parallel sequencing, an extremely powerful method for microRNA (miRNA) discovery, has not yet been applied to bacterial transcriptomes. Here we present sRNA-Seq, an unbiased method that allows for interrogation of the entire small, non-coding RNA (sRNA) repertoire in any prokaryotic or eukaryotic organism. This method includes a novel treatment that depletes total RNA fractions of highly abundant tRNAs and small subunit rRNA, thereby enriching the starting pool for sRNA transcripts with novel functionality. As a proof-of-principle, we applied sRNA-Seq to the human pathogen Vibrio cholerae. Our results provide information, at unprecedented depth, on the complexity of the sRNA component of a bacterial transcriptome. From 407 039 sequence reads, all 20 known V. cholerae sRNAs, 500 new, putative intergenic sRNAs and 127 putative antisense sRNAs were identified in a limited number of growth conditions examined. In addition, characterization of a subset of the newly identified transcripts led to the identification of a novel sRNA regulator of carbon metabolism. Collectively, these results strongly suggest that the number of sRNAs in bacteria has been greatly underestimated and that future efforts to analyze bacterial transcriptomes will benefit from direct cloning and parallel sequencing experiments aided by 5S/tRNA depletion.

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Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing: a randomized trial.
Amina Abdelaal, Hassan Abd El-Ghaffar, Mohammad Hosam Eldeen Zaghloul, Noha El mashad, Ehab Badran, Amal Fathy.
Annals of Clinical Microbiology and Antimicrobials 8, 4 (2009) | doi:10.1186/1476-0711-8-4 | PMID:19183459
Annals of Clinical Microbiology and Antimicrobials 8, 5 (2009) | doi:10.1186/1476-0711-8-5 | PMID:19220891 (Correction)

Background
Tuberculosis is a growing international health concern. It is the biggest killer among the infectious diseases in the world today. Early detection of drug resistance allows starting of an appropriate treatment. Resistance to drugs is due to particular genomic mutations in specific genes of Mycobacterium tuberculosis(MTB). The aim of this study was to identify the presence of Isoniazid (INH) and Rifampicin(RIF) drug resistance in new and previously treated tuberculosis (TB) cases using DNA sequencing.

Methods
This study was carried out on 153 tuberculous patients with positive Bactec 460 culture for acid fast bacilli.

Results
Of the 153 patients, 105 (68.6%) were new cases and 48 (31.4%) were previously treated cases.Drug susceptibility testing on Bactec revealed 50 resistant cases for one or more of the first line antituberculous. Genotypic analysis was done only for rifampicin resistant specimens (23 cases) and INH resistant specimens (26 cases) to detect mutations responsible for drug resistance by PCR amplification of rpoB gene for rifampicin resistant cases and KatG gene for isoniazid resistant cases. Finally, DNA sequencing was done for detection of mutation within rpoB and katG genes. Genotypic analysis of RIF resistant cases revealed that 20/23 cases (86.9%) of RIF resistance were having rpoB gene mutation versus 3 cases (13.1%) having no mutation with a high statistical significant difference between them ( P<0.001). Direct sequencing of gene revealed point mutation in 24/26 (92.3%) and the remaining 2 /26 (7.7%) had wild type katG i.e. no evidence of mutation with a high statistical significant difference between them ( P<0.001). Conclusions
We can conclude that rifampicin resistance could be used as a useful surrogate marker for estimation of multidrug resistance. In addition, Genotypic method was superior to that of the traditional phenotypic method which is time-consuming taking several weeks or longer.

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Massively parallel sequencing identifies the gene Megf8 with ENU-induced mutation causing heterotaxy.
Zhen Zhang, Deanne Alpert, Richard Francis, Bishwanath Chatterjee, Qing Yu, Terry Tansey, Steven L. Sabol, Cheng Cui, Yongli Bai, Maxim Koriabine, Yuko Yoshinaga, Jan-Fang Cheng, Feng Chen, Joel Martin, Wendy Schackwitz, Teresa M. Gunn, Kenneth L. Kramer, Pieter J. De Jong, Len A. Pennacchio, Cecilia W. Lo.
PNAS 106, 3219-3224 (2009) | doi:10.1073/pnas.0813400106 | PMID:19218456

Forward genetic screens with ENU (N-ethyl-N-nitrosourea) mutagenesis can facilitate gene discovery, but mutation identification is often difficult. We present the first study in which an ENU- induced mutation was identified by massively parallel DNA sequencing. This mutation causes heterotaxy and complex congenital heart defects and was mapped to a 2.2-Mb interval on mouse chromosome 7. Massively parallel sequencing of the entire 2.2-Mb interval identified 2 single-base substitutions, one in an intergenic region and a second causing replacement of a highly conserved cysteine with arginine (C193R) in the gene Megf8. Megf8 is evolutionarily conserved from human to fruit fly, and is observed to be ubiquitously expressed. Morpholino knockdown of Megf8 in zebrafish embryos resulted in a high incidence of heterotaxy, indicating a conserved role in laterality specification. Megf8^C193R mouse mutants show normal breaking of symmetry at the node, but Nodal signaling failed to be propagated to the left lateral plate mesoderm. Videomicroscopy showed nodal cilia motility, which is required for left–right patterning, is unaffected. Although this protein is predicted to have receptor function based on its amino acid sequence, surprisingly confocal imaging showed it is translocated into the nucleus, where it is colocalized with Gfi1b and Baf60C, two proteins involved in chromatin remodeling. Overall, through the recovery of an ENU-induced mutation, we uncovered Megf8 as an essential regulator of left–right patterning.