PCR Errors Research Articles

Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacerrDNAregion in fungi

The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular investigations of environmental samples. In this study 454 amplicon pyrosequencing of the ITS1 region was applied to 99 phylogenetically diverse axenic single-spore cultures of fungi (Dikarya: Ascomycota and Basidiomycota) to investigate levels of intragenomic variation. Three species (one Basidiomycota and two Ascomycota), in addition to a positive control species known to contain ITS paralogs, displayed levels of molecular variation indicative of intragenomic variation; taxon inflation due to presumed intragenomic variation was ≈9%. Intragenomic variability in the ITS region appears to be widespread but relatively rare in fungi (≈3–5% of species investigated in this study), suggesting this problem may have minor impacts on species richness estimates relative to PCR and/or pyrosequencing errors. Our results indicate that 454 amplicon pyrosequencing represents a powerful tool for investigating levels of ITS intragenomic variability across taxa, which may be valuable for better understanding the fundamental mechanisms underlying concerted evolution of repetitive DNA regions.

Abstract 5137: Implementing an automated clinical next generation sequencing analysis pipeline - assuring accurate and robust results.

Abstract Introduction: There is unmet clinical need for automated data processing of deep sequencing in heterogeneous sample populations in molecular oncology specimens. We have developed clinical assays using Next Generation Sequencing (NGS) for deep sequencing in both solid tumor and hematological indications. We have targeted five genes (ASXL1, RUNX1, EZH2, ETV6, and TP53) involved in myelodysplastic syndrome (MDS) and three genes (BRAF, c-KIT, NRAS) in late stage melanoma. We designed and implemented a data processing pipeline that integrates novel and commercial analysis software to provide automated rules-based filtering and annotation of variants. Methods: We used a targeted resequencing approach with the Fluidigm Access Array system for library creation and molecular barcoding for sample multiplexing. Samples were processed in duplicate to filter out potential false positives. Libraries were sequenced using the Ion Torrent Personal Genome Machine (PGM) (Life Technologies, Carlsbad, CA) and the Illumina MiSeq (Illumina Inc., San Diego, CA) systems. Raw data in FastQ format was preprocessed using Windows batch commands and Perl programs that were integrated with NextGENe (SoftGenetics, State College, PA) analysis software for quality trimming, alignment and variant calling against NCBI curated references. Variant calls were compared between duplicate samples and annotated using a separate Perl program that queried databases containing known germline and somatic variants. Results: Quality control was implemented by trimming all downstream reads that contained specified Q-scores (Phred, Q16 or above) as well as requiring a minimum coverage depth of 500X per exon. Only variant calls confirmed at greater than 5% in both duplicate samples and with a balanced ratio of forward and reverse reads were considered reportable in a clinical setting. Variants appearing in only one of the duplicates were consistent with systematic errors. Variant calls were annotated with existing database information or were saved in a database of novel mutations by the annotation pipeline. The pipeline removes all systematic errors in known cell line samples by filtering metrics. Conclusions: We have developed a robust and automated pipeline for NGS data analysis in the clinical lab that is independent of sequencing platform. The implementation of an automated processing pipeline increases the efficiency and consistency of analysis. Hands-on time for data analysis has been decreased from 30 minutes per sample to less than 5 minutes per sample. Carrying duplicates through the entire multiplexing and sequencing process also helps reduce test based false-positive errors (e.g. PCR errors) while robust analytical metrics remove other sources of error (e.g. strand bias). We have found an average of 6-10 false positive calls in both sample types, all of which were filtered out by the pipeline presented here. Citation Format: Adam Baer, Yu Xia, Todd Holscher, Robyn Loper, Matthew J. McGinniss, Richard Tseng, Julie Kines, Jonathan Diver, Jason Christiansen. Implementing an automated clinical next generation sequencing analysis pipeline - assuring accurate and robust results. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5137. doi:10.1158/1538-7445.AM2013-5137

PyroClean: Denoising Pyrosequences from Protein-Coding Amplicons for the Recovery of Interspecific and Intraspecific Genetic Variation

High-throughput parallel sequencing is a powerful tool for the quantification of microbial diversity through the amplification of nuclear ribosomal gene regions. Recent work has extended this approach to the quantification of diversity within otherwise difficult-to-study metazoan groups. However, nuclear ribosomal genes present both analytical challenges and practical limitations that are a consequence of the mutational properties of nuclear ribosomal genes. Here we exploit useful properties of protein-coding genes for cross-species amplification and denoising of 454 flowgrams. We first use experimental mixtures of species from the class Collembola to amplify and pyrosequence the 5′ region of the COI barcode, and we implement a new algorithm called PyroClean for the denoising of Roche GS FLX pyrosequences. Using parameter values from the analysis of experimental mixtures, we then analyse two communities sampled from field sites on the island of Tenerife. Cross-species amplification success of target mitochondrial sequences in experimental species mixtures is high; however, there is little relationship between template DNA concentrations and pyrosequencing read abundance. Homopolymer error correction and filtering against a consensus reference sequence reduced the volume of unique sequences to approximately 5% of the original unique raw reads. Filtering of remaining non-target sequences attributed to PCR error, sequencing error, or numts further reduced unique sequence volume to 0.8% of the original raw reads. PyroClean reduces or eliminates the need for an additional, time-consuming step to cluster reads into Operational Taxonomic Units, which facilitates the detection of intraspecific DNA sequence variation. PyroCleaned sequence data from field sites in Tenerife demonstrate the utility of our approach for quantifying evolutionary diversity and its spatial structure. Comparison of our sequence data to public databases reveals that we are able to successfully recover both interspecific and intraspecific sequence diversity.

Huge Overlap of Individual TCR Beta Repertoires

Huge Overlap of Individual TCR Beta Repertoires

Time Course Studies of the Progeny of Barcoded CD34+ Human Cord Blood Cells Generated in Transplanted NOD/SCID-IL2Rγ−/− Mice Unveil the Clonal Dynamics of Short Term, Intermediate Term and Long Term Repopulating Cells

AbstractAbstract 28Hematopoietic stem cells (HSC) exhibit heterogeneity in self-renewal and differentiation activity, but the extent to which this is intrinsically determined and extrinsically regulated is still poorly understood. In the mouse, purities of HSCs can now be achieved to allow such questions to be addressed directly. Interestingly, tracking the outputs of large numbers of serial transplantable clones produced from single-cell transplants, or the clonal progenies of vector-marked/barcoded cells indicate the existence in mice of 2 subsets of HSCs with durable self-renewal ability. These 2 subsets are characterized by distinct lineage output programs that are maintained through the many HSC self-renewal divisions required to serially propagate a clone in vivo. To begin to ask whether similar subsets of human HSCs exist, we have created a diverse lentiviral library encoding an estimated >105 different barcode sequences and GFP, and then used this library to track the in vivo clonal outputs of transduced human CD34+ cord blood cells in xenografted mice. For this experiment, CD34+ cells isolated immunomagnetically to a purity of >80% were exposed to virus for 6 hours in the presence of growth factors and then immediately injected intravenously into 2 sublethally irradiated NOD/SCID-IL2Rγ−/− mice (1.2 × 105 cells per mouse; 30% GFP+ cells after 3 days in vitro). Different subsets of human cells were then isolated by FACS from immunostained bone marrow cells aspirated sequentially from the femurs of the mice at intervals from 4–27 weeks post-transplant and the identity, number and size of clones in each established by next generation sequencing of barcoded amplicons derived from each sample. To identify barcodes arising from PCR and sequencing errors and calibrate clone sizes, we included 3 controls of 20, 100 and 500 cells with a known barcode at each datapoint. The data from these controls allowed a threshold of 20 cells per clone to be established with >95% confidence. We then compared the representation of clones among all samples from each mouse to derive the number and size of all clones detected, assuming a mouse contains 2×108 bone marrow cells. This analysis revealed a total of 154 uniquely barcoded clones containing up to 2×108 human hematopoietic cells in the 2 mice (8–30 × 106 in one and 4–165 × 106 in the other at any single time point). Analysis of the representation of each clone over time showed successive waves of repopulation from different clones with lineage output profiles consistent with those obtained by transplanting separate fractions of CD34+ cord blood cells distinguished by their surface phenotypes. Specifically, we detected 50 clones (32% of all clones) that were not sustained at detectable levels beyond 9 weeks post-transplantation and were characterized by robust myeloid differentiation with variable B cell outputs at 4 weeks. Another 30 clones (19%) showed significant but also transient outputs of either or both the myeloid and B cell lineage, disappearing between week 9 and 16 post-transplant. Mature cell output was detected from a total of 74 clones (48%) at the 27 week time point, among which 36 (23%) were not evident during the first 4 months post-transplant. These late-appearing clones were mostly small (contributing up to 3 × 105 total hematopoietic cells at week 27) and made a significantly higher contribution to the total human myeloid population than to the total human B cell population. Notably, the 12 long term clones that showed robust mature cell output detectable in all 3 sites sampled at week 27 when the mice were sacrificed (left leg vs right leg vs pelvis) contained both myeloid and lymphoid cells but with large (>100-fold) variations in their representation in the 3 different sites. This latter finding suggests less trafficking of human cells between sites than expected from parabiotic mouse experiments or substantial differences in the differentiation control exerted in different locations. Additionally, from one of the mice, we obtained the first direct evidence of a large output of human T cells (>9 × 106) that was part of a long term multi-lineage clone detectable at 27 weeks post-transplant. This first use of a barcoding strategy to analyze the clonal dynamics of normal human CD34+ cells with in vivo repopulating activity demonstrates the power of this approach to analyze their lineage outputs and sets the stage for novel applications to expanded and transformed populations. Disclosures:No relevant conflicts of interest to declare.

Denoising PCR-amplified metagenome data

BackgroundPCR amplification and high-throughput sequencing theoretically enable the characterization of the finest-scale diversity in natural microbial and viral populations, but each of these methods introduces random errors that are difficult to distinguish from genuine biological diversity. Several approaches have been proposed to denoise these data but lack either speed or accuracy.ResultsWe introduce a new denoising algorithm that we call DADA (Divisive Amplicon Denoising Algorithm). Without training data, DADA infers both the sample genotypes and error parameters that produced a metagenome data set. We demonstrate performance on control data sequenced on Roche’s 454 platform, and compare the results to the most accurate denoising software currently available, AmpliconNoise.ConclusionsDADA is more accurate and over an order of magnitude faster than AmpliconNoise. It eliminates the need for training data to establish error parameters, fully utilizes sequence-abundance information, and enables inclusion of context-dependent PCR error rates. It should be readily extensible to other sequencing platforms such as Illumina.

Next generation sequencing for TCR repertoire profiling: Platform‐specific features and correction algorithms

The TCR repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next generation sequencing (NGS) is becoming a powerful tool for deep TCR profiling; yet, questions abound regarding the methodological approaches for sample preparation and correct data interpretation. Accumulated PCR and sequencing errors along with library preparation bottlenecks and uneven PCR efficiencies lead to information loss, biased quantification, and generation of huge artificial TCR diversity. Here, we compare Illumina, 454, and Ion Torrent platforms for individual TCR profiling, evaluate the rate and character of errors, and propose advanced platform-specific algorithms to correct massive sequencing data. These developments are applicable to a wide variety of next generation sequencing applications. We demonstrate that advanced correction allows the removal of the majority of artificial TCR diversity with concomitant rescue of most of the sequencing information. Thus, this correction enhances the accuracy of clonotype identification and quantification as well as overall TCR diversity measurements.

Identification of Amino Acids Important for Substrate Specificity in Sucrose Transporters Using Gene Shuffling

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.

Detection of ultra-rare mutations by next-generation sequencing

Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of ~1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when "deep sequencing" genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage. We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.

Prevalence of canine haemotropic mycoplasma infections in Sydney, Australia

Haemotropic mycoplasmas (haemoplasmas) are epierythrocytic, wall-less bacteria that cannot be cultivated in vitro (Messick 2003). Two species have been identified in dogs: Mycoplasma haemocanis (Mhc) and ‘ Candidatus Mycoplasma haematoparvum’...

Uninformative polymorphisms bias genome scans for signatures of selection

BackgroundWith the establishment of high-throughput sequencing technologies and new methods for rapid and extensive single nucleotide (SNP) discovery, marker-based genome scans in search of signatures of divergent selection between populations occupying ecologically distinct environments are becoming increasingly popular.Methods and ResultsOn the basis of genome-wide SNP marker data generated by RAD sequencing of lake and stream stickleback populations, we show that the outcome of such studies can be systematically biased if markers with a low minor allele frequency are included in the analysis. The reason is that these ‘uninformative’ polymorphisms lack the adequate potential to capture signatures of drift and hitchhiking, the focal processes in ecological genome scans. Bias associated with uninformative polymorphisms is not eliminated by just avoiding technical artifacts in the data (PCR and sequencing errors), as a high proportion of SNPs with a low minor allele frequency is a general biological feature of natural populations.ConclusionsWe suggest that uninformative markers should be excluded from genome scans based on empirical criteria derived from careful inspection of the data, and that these criteria should be reported explicitly. Together, this should increase the quality and comparability of genome scans, and hence promote our understanding of the processes driving genomic differentiation.

Degenerate Primer IDs and the Birthday Problem

In PNAS, Jabara et al. (1) proposed an important approach to correct for recombination, allelic skewing, misincorporations, and sequencing errors in deep sequencing technologies. Their approach relies on each viral RNA template receiving a unique, randomly generated “Primer ID.” They were thus able to quantify template resampling by counting the repeated detection of each unique ID. Furthermore, they were able to exploit template resampling to correct for PCR errors, sequencing errors, and allelic skewing by only retaining consensus sequences where at least three reads shared a Primer ID.

Bioinformatic challenges for DNA metabarcoding of plants and animals

Almost all empirical studies in ecology have to identify the species involved in the ecological process under examination. DNA metabarcoding, which couples the principles of DNA barcoding with next generation sequencing technology, provides an opportunity to easily produce large amounts of data on biodiversity. Microbiologists have long used metabarcoding approaches, but use of this technique in the assessment of biodiversity in plant and animal communities is under-explored. Despite its relationship with DNA barcoding, several unique features of DNA metabarcoding justify the development of specific data analysis methodologies. In this review, we describe the bioinformatics tools available for DNA metabarcoding of plants and animals, and we revisit others developed for DNA barcoding or microbial metabarcoding. We also discuss the principles and associated tools for evaluating and comparing DNA barcodes in the context of DNA metabarcoding, for designing new custom-made barcodes adapted to specific ecological question, for dealing with PCR and sequencing errors, and for inferring taxonomical data from sequences.

Chlamydia Psittaci negative MALT Lymphomas of the Ocular Adnexa Are Characterized by Biased Use of the VH4 Family and Evidence for Antigen Selection

Abstract 2644Mucosa-associated lymphoid tissue (MALT) lymphomas are the most common lymphomas in the ocular adnexa. Similar to extranodular MALT lymphomas in other tissues, ocular adnexa MALT lymphomas (OAMALTL) are frequently characterized by an indolent clinical course and often remain localized for many years. While chronic infection (e. g. Helicobacter pylori) or autoimmune diseases (e.g. Hashimoto’s thyroiditis and Sjogren’s syndrome) frequently precede and predispose patients to develop MALT lymphomas in the stomach, thyroid and salivary glands, respectively, the etiology and pathogenesis of OAMALTL are still controversial. An Italian group and investigators from several other geographic regions demonstrated Chlamydia psittaci (C. psittaci) DNA present in OAMALTL, suggesting that this pathogen may be implicated in the development of these lymphomas. However, similar studies performed by us and other investigators on US-based patients have failed to corroborate this finding. Furthermore, DNA from other bacteria was also not detected by us, thus not supporting a bacterial etiology in US-based patients. Since non-bacterial antigens may predispose patients to OAMALTL, we have examined immunoglubulin (Ig) heavy chain variable region (VH) usage and mutations in the largest to date cohort of C psittaci negative OAMALTL originating from a single institution. DNA was extracted by standard techniques from 68 fresh OAMALTL tissues and used for direct PCR or PCR followed by cloning and sequencing using family specific VH leader and JH primers. To control for potential PCR error, all patient samples were evaluated by 2 independent PCR reactions and sequencing and only cases with identical CDR3 regions in the independent PCR runs were defined as clonal. Using this definition, clonal rearranged VHDJH sequences were identified in 44 (64.7%) tumors originating from the orbit (19), conjunctivae (19) and lacrimal gland (6). Forty seven clonal VH gene sequences were detected (3 of the patients had 2 clonal sequences) with 46 potentially functional and 1 harboring an out-of-frame junction with a stop codon. In 14 (31.8%) cases the PCR product could be sequenced directly, whereas in 30 (68.2%) cases PCR amplicons had to be subcloned to identify the VH gene. The 46 potentially functional VH were derived from 4 of the 7 human VH gene families with the following distribution: VH1, 13.1%; VH2, 2.2%; VH3, 39.1%; VH4, 45.6%, demonstrating a biased overrepresentation of the VH4 gene family (p=0.001). The most frequently encountered genes were VH4-34 (n=8), VH3-30 (n=6), VH3-23 (n=5) and VH4-30 (n=4). VH4-34 represented 17% of all the potentially functional VH genes identified in this study in contrast to its usage in 4–7% in adult peripheral B lymphocytes. Only 3 VH gene sequences were unmutated. The average percent homology to the germ line sequence in the 43 functional mutated sequences was 93.2% (range 71.5–99.6). Multinomial algorithm for antigen selection revealed evidence for positive selection in the CDR in 15 sequences, negative selection in the FR in 17 sequences, and selection in both CDR and FR (in the same sequence) in 7 sequences. Selection analysis using the focused binomial test demonstrated evidence of selection in the FR in 12 sequences and in the CDR in only 4 sequences. Selection was detected in 5 of the 8 functional VH4-34 sequences. Analysis of the tumor-derived CDR3 sequences revealed low similarity and an absence of stereotyped sequences with no homology to antibacterial and other previously published antibodies. The average CDR3 isoelectric point was 5.91±1.89 (SD). The average CDR3 length was 15.73±13.62 (SD) amino acids, with 19 sequences harboring 15–19 amino acids and 7 CDR3 sequences longer than 19 amino acids. Intraclonal variation was assessed by extensive molecular cloning in 8 potentially functional VH gene isolates from 8 randomly selected tumor specimens. Confirmed ongoing mutations were detected in the 6 of the 8 analyzed sequences. Overall our findings demonstrate that C. psittaci negative OAMALTL exhibit biased usage of VH families and genes with evidence for intraclonal heterogeneity and antigen selection in multiple tumors, implicating immunological stimulation in the pathogenesis of these lymphomas. The nature of the antigens potentially playing role in these processes is currently unknown and requires further studies. Disclosures:No relevant conflicts of interest to declare.

Microarray Generation of Thousand-Member Oligonucleotide Libraries

The ability to efficiently and economically generate libraries of defined pieces of DNA would have a myriad of applications, not least in the area of defined or directed sequencing and synthetic biology, but also in applications associated with encoding and tagging. In this manuscript DNA microarrays were used to allow the linear amplification of immobilized DNA sequences from the array followed by PCR amplification. Arrays of increasing sophistication (1, 10, 3,875, 10,000 defined sequences) were used to validate the process, with sequences verified by selective hybridization to a complementary DNA microarray and DNA sequencing, which demonstrated a PCR error rate of 9.7×10−3/site/duplication. This technique offers an economical and efficient way of producing specific DNA libraries of hundreds to thousands of members with the DNA-arrays being used as “factories” allowing specific DNA oligonucleotide pools to be generated. We also found substantial variance observed between the sequence frequencies found via Solexa sequencing and microarray analysis, highlighting the care needed in the interpretation of profiling data.

Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates

Different hepatitis B virus (HBV) genotypes and variants are associated with different clinical outcomes and/or response to antiviral therapy, yet the comparison of the in vitro replication capacity of a large number of clinical isolates remains technically challenging and time-consuming. Although the full-length HBV genome can be amplified from high-titer blood samples by PCR using High Fidelity(plus) DNA polymerase and primers targeting the conserved precore region, the HBV clones thus generated are replication deficient due to the inability to generate the terminally redundant pregenomic RNA essential for genome replication. The transfection experiment is further complicated by PCR errors and the presence of viral quasispecies. A previous study found that the precise removal of non-HBV sequence by SapI digestion led to HBV replication in transfected cells, possibly due to low-level genome circularization by a cellular enzyme. We released HBV genome from the cloning vector using BspQI, an inexpensive isoschizomer of SapI, and increased the efficiency of genome replication by an extra step of in vitro DNA ligation. The uncut plasmid DNA can be used for transfection if the sole purpose is to study envelope protein expression. We found significant PCR errors associated with the High Fidelity(plus) DNA polymerase, which could be greatly diminished using Phusion DNA polymerase or masked by the use of a clone pool. The reduced PCR error and modified enzymatic steps prior to transfection should facilitate a more widespread functional characterization of clinical HBV isolates, while the clone pool approach is useful for samples with significant sequence heterogeneity.

Removal of PCR Error Products and Unincorporated Primers by Metal-Chelate Affinity Chromatography

Immobilized Metal Affinity Chromatography (IMAC) has been used for decades to purify proteins on the basis of amino acid content, especially surface-exposed histidines and “histidine tags” genetically added to recombinant proteins. We and others have extended the use of IMAC to purification of nucleic acids via interactions with the nucleotide bases, especially purines, of single-stranded RNA and DNA. We also have demonstrated the purification of plasmid DNA from contaminating genomic DNA by IMAC capture of selectively-denatured genomic DNA. Here we describe an efficient method of purifying PCR products by specifically removing error products, excess primers, and unincorporated dNTPs from PCR product mixtures using flow-through metal-chelate affinity adsorption. By flowing a PCR product mixture through a Cu2+-iminodiacetic acid (IDA) agarose spin column, 94–99% of the dNTPs and nearly all the primers can be removed. Many of the error products commonly formed by Taq polymerase also are removed. Sequencing of the IMAC-processed PCR product gave base-calling accuracy comparable to that obtained with a commercial PCR product purification method. The results show that IMAC matrices (specifically Cu2+-IDA agarose) can be used for the purification of PCR products. Due to the generality of the base-specific mechanism of adsorption, IMAC matrices may also be used in the purification of oligonucleotides, cDNA, mRNA and micro RNAs.

Variability in the IGS1 region of Rhodocollybia laulaha: is it allelic, genomic, or artificial?

We tested the nuclear ribosomal intergenic spacer 1 ( IGS1) to evaluate: 1) the utility of this gene region in the study of intra-lineage variation in the Hawaiian mushroom Rhodocollybia laulaha ( Basidiomycota, Marasmiaceae); 2) the possibility of PCR-mediated artifacts from multiple cloned IGS1 inserts for five of the nine specimens included; and 3) the assignment of individual specimens to specific haplotype group genotypes. The IGS1 was sequenced for nine specimens of R. laulaha and multiple cloned copies of the IGS1 were sequenced for five of those nine to compare intra-specimen variation ( i.e. among paralogous copies in a single genome) and inter-specimen variation ( i.e. among different specimens). Three haplotype groups were identified with 25 of 510 aligned base pairs being variable (17 substitutions and eight indels). The observed variation could be due to the presence of various alleles of the IGS1 locus and/or variation among paralogous copies within a single genome. Issues associated with PCR error including artificial recombination and/or Taq error and the potential inefficiency of concerted evolution are considered as possible agents of variation.

Genetic Analysis of a UNAIDS HIV Type 1 from Brazil Revealed an Unexpected Recombination Pattern

Genetic Analysis of a UNAIDS HIV Type 1 from Brazil Revealed an Unexpected Recombination Pattern

Metallothionein-1 Isoforms and Vimentin Are Direct PU.1 Downstream Target Genes in Leukemia Cells

PU.1 is a key transcription factor for hematopoiesis and plays important roles in various hematological malignancies. To clarify the molecular function of PU.1, we initially tried to identify bona fide target genes regulated by PU.1. Dual microarrays were employed for this study to compare PU.1-knockdown K562 cells (K562PU.1KD) stably expressing PU.1 short inhibitory RNAs versus control cells and PU.1-overexpressing K562 cells (K562PU.1OE) versus control cells. In these analyses, we found that several genes, including metallothionein (MT)-1 isoforms (MT-1G and MT-1A) and vimentin (VIM), were markedly induced while Jun dimerization protein (JDP) 2 was suppressed in K562PU.1KD cells. Furthermore, the mRNA expressions of the MT-1 and VIM genes were inversely correlated and the mRNA expression of JDP2 was positively correlated with PU.1 mRNA expression in 43 primary acute myeloid leukemia specimens (MT-1G: R = -0.50, p < 0.001; MT-1A: R = -0.58, p < 0.0005; VIM: R = -0.39, p < 0.01; and JDP2: R = 0.30, p < 0.05). Next, we analyzed the regulation of the MT-1 and VIM genes. We observed increased associations of acetylated histones H3 and H4 with the promoters of these genes in K562PU.1KD cells. Sequence analyses of the regions approximately 1 kb upstream from the transcription start sites of these genes revealed numerous CpG sites, which are potential targets for DNA methylation. Chromatin immunoprecipitation assays revealed that methyl CpG-binding protein 2 (MeCP2) and PU.1 bound to the CpG-rich regions in the MT-1 and VIM promoters. Bisulfite sequencing analyses of the PU.1-bound regions of these promoters revealed that the proportions of methylated CpG sites were tightly related to the PU.1 expression levels.