Next-generation sequencing of HIV-1 RNA genomes: determination of error rates and minimizing artificial recombination.

Next-generation sequencing (NGS) is a valuable tool for the detection and quantification of HIV-1 variants in vivo. However, these technologies require detailed characterization and control of artificially induced errors to be applicable for accurate haplotype reconstruction. To investigate the occurrence of substitutions, insertions, and deletions at the individual steps of RT-PCR and NGS, 454 pyrosequencing was performed on amplified and non-amplified HIV-1 genomes. Artificial recombination was explored by mixing five different HIV-1 clonal strains (5-virus-mix) and applying different RT-PCR conditions followed by 454 pyrosequencing. Error rates ranged from 0.04-0.66% and were similar in amplified and non-amplified samples. Discrepancies were observed between forward and reverse reads, indicating that most errors were introduced during the pyrosequencing step. Using the 5-virus-mix, non-optimized, standard RT-PCR conditions introduced artificial recombinants in a fraction of at least 30% of the reads that subsequently led to an underestimation of true haplotype frequencies. We minimized the fraction of recombinants down to 0.9-2.6% by optimized, artifact-reducing RT-PCR conditions. This approach enabled correct haplotype reconstruction and frequency estimations consistent with reference data obtained by single genome amplification. RT-PCR conditions are crucial for correct frequency estimation and analysis of haplotypes in heterogeneous virus populations. We developed an RT-PCR procedure to generate NGS data useful for reliable haplotype reconstruction and quantification.

Role of {\it ERCC1\/} in DNA repair and genetic recombination

The ERCC1 (Excision Repair Cross-Complementing-1) gene is the presumptive mammalian homolog of the Saccharomyces cerevisiae RAD10 gene. In mammalian NER, the Ercc1/XpF complex functions as an endonuclease that specifically recognizes 5$\sp\prime$ double-strand-3$\sp\prime$ single-strand structures. In yeast, the analogous function is performed by the Rad1/Rad10 complex. These observations and the conservation of amino acid homology between the Rad1 and XpF and the Rad10 and Ercc1 proteins has led to a general assumption of functional homology between these genes.^ In addition to NER, the Rad1/Rad10 endonuclease complex is also required in certain specialized mitotic recombination pathways in yeast. However, a similiar requirement for the endonuclease function of the Ercc1/XpF complex during genetic recombination in mammalian cells has not been directly demonstrated. The experiments performed in these studies were designed to determine if ERCC1 deficiency would produce recombination-deficient phenotypes in CHO cells similar to those observed in RAD10 deletion mutants, including: (1) decreased single-reciprocal exchange recombination, and (2) inability to process 5$\sp\prime$ sequence heterology in recombination intermediates.^ Specifically, these studies describe: (1) The isolation and characterization of the ERCC1 locus of Chinese hamster ovary cells; (2) The production of an ERCC1 null mutant cell line by targeted knock-out of the endogenous ERCC1 gene in a Chinese hamster ovary cell line, CHO-ATS49tg, which contains an endogenous locus, APRT, suitable as a chromosomal target for homologous recombination; (3) The characterization of mutant ERCC1 alleles from a panel of Chinese hamster ovary cell ERCC1 mutants derived by conventional mutagenesis; (4) An investigation of the effects of ERCC1 mutation on mitotic recombination through targeting of the APRT locus in an ERCC1 null background.^ The results of these studies strongly suggest that the role of ERCC1 in homologous recombination in mammalian cells is analogous to that of the yeast RAD10 gene. ^

Methods for preparing and counting biochemical varves

Four different preparation and counting methods for biochemical varves were compared in order to assess counting errors and to standardize these techniques. The properties of two embedding methods, namely the shock-freeze, freeze-dry and the water-acetone-epoxy-exchange method, are discussed. Varve counts were carried out on fresh sediment and on sediment thin-sections, on the latter by manual and by automated counting using image-analysis software. Counting on fresh sediment and using image-analysis generally underestimated the number of varves, especially in sections with inconspicuous varves. A comparison between multiple varve counts carried out by a single analyst and different analysts showed no significant differences in the mean varve counts.

Characterization of template switching and recombination during Moloney murine leukemia virus replication

Retroviruses uniquely co-package two copies of their genomic RNA within each virion. The two copies are used as templates for synthesis of the proviral DNA during the process of reverse transcription. Two template switches are required to complete retroviral DNA synthesis by the retroviral enzyme, reverse transcriptase. With two RNA genomes present in the virion, reverse transcriptase can make template switches utilizing only one of the RNA templates (intramolecular) or utilizing both RNA templates (intermolecular) during the process of reverse transcription. The results presented in this study show that during a single cycle of Moloney murine leukemia virus replication, both nonrecombinant and recombinant proviruses predominantly underwent intramolecular minus- and plus-strand transfers during the process of reverse transcription. This is the first study to examine the nature of the required template switches occurring during MLV replication and these results support the previous findings for SNV, and the hypothesis that the required template switches are ordered events. This study also determined rates for deletion and a rate of recombination for a single cycle of MLV replication. The rates reported here are comparable to the rates previously reported for both SNV and MLV. ^

Petrology and geochemistry of silicified upper Miocene chalk at DSDP Site 69-504

Chert, Porcellanite, and other silicified rocks formed in response to high heat flow in the lower 50 meters of 275 meters of sediments at Deep Sea Drilling Project Site 504, Costa Rica Rift. Chert and Porcellanite partly or completely replaced upper Miocene chalk and limestone. Silicified rock occurs as nodules, laminae, stringers, and casts of burrows, and consists of quartz and opal-CT in varying amounts, associated with secondary calcite. The secondary silica was derived from dissolution of opal-A (biogenic silica), mostly diatom frustules and radiolarian tests. Temperature data obtained at the site indicate that transformation of opal-A to opal-CT began at about 50°C, and transformation from opal-CT to quartz at about 55°C. Quartz is most abundant close to basement basalts. These silica transformations occurred over the past 1 m.y., and took place so rapidly that there was incomplete ordering of opal-CT before transformation to quartz; opal-CT formed initially with an uncommonly wide d spacing. Quartz shows poor crystallinity. Chemical data show that the extensively silicified rocks consist of over 96% SiO2; in these rocks, minor and trace elements decreased greatly, except for boron, which increased. Low Al2O3 and TiO2 contents in all studied rocks preclude the presence of significant volcanic or terrigenous detritus. Mn content increases with depth, perhaps reflecting contributions from basalts or hydrothermal solutions. Comparisons with cherts from oceanic plateaus in the central Pacific point to a more purely biogenic host sediment for the Costa Rica Rift cherts, more rapid precipitation of quartz, and formation nearer a spreading center. Despite being closer to continental sources of ash and terrigenous detritus, Costa Rica Rift cherts have lower Al2O3, Fe2O3, and Mn concentrations.

Automated Tools for Layer Recognition (BMPix) and Counting (PEAK)

We present tools for rapid and quantitative detection of sediment lamination. The BMPix tool extracts color and gray-scale curves from images at pixel resolution. The PEAK tool uses the gray-scale curve and performs, for the first time, fully automated counting of laminae based on three methods. The maximum count algorithm counts every bright peak of a couplet of two laminae (annual resolution) in a smoothed curve. The zero-crossing algorithm counts every positive and negative halfway-passage of the curve through a wide moving average, separating the record into bright and dark intervals (seasonal resolution). The same is true for the frequency truncation method, which uses Fourier transformation to decompose the curve into its frequency components before counting positive and negative passages. We applied the new methods successfully to tree rings, to well-dated and already manually counted marine varves from Saanich Inlet, and to marine laminae from the Antarctic continental margin. In combination with AMS14C dating, we found convincing evidence that laminations in Weddell Sea sites represent varves, deposited continuously over several millennia during the last glacial maximum. The new tools offer several advantages over previous methods. The counting procedures are based on a moving average generated from gray-scale curves instead of manual counting. Hence, results are highly objective and rely on reproducible mathematical criteria. Also, the PEAK tool measures the thickness of each year or season. Since all information required is displayed graphically, interactive optimization of the counting algorithms can be achieved quickly and conveniently.