Quantitative Bias in Illumina TruSeq and a Novel Post Amplification Barcoding Strategy for Multiplexed DNA and Small RNA Deep Sequencing

Here we demonstrate a method for unbiased multiplexed deep sequencing of RNA and DNA libraries using a novel, efficient and adaptable barcoding strategy called Post Amplification Ligation-Mediated (PALM). PALM barcoding is performed as the very last step of library preparation, eliminating a potential barcode-induced bias and allowing the flexibility to synthesize as many barcodes as needed. We sequenced PALM barcoded micro RNA (miRNA) and DNA reference samples and evaluated the quantitative barcode-induced bias in comparison to the same reference samples prepared using the Illumina TruSeq barcoding strategy. The Illumina TruSeq small RNA strategy introduces the barcode during the PCR step using differentially barcoded primers, while the TruSeq DNA strategy introduces the barcode before the PCR step by ligation of differentially barcoded adaptors. Results show virtually no bias between the differentially barcoded miRNA and DNA samples, both for the PALM and the TruSeq sample preparation methods. We also multiplexed miRNA reference samples using a pre-PCR barcode ligation. This barcoding strategy results in significant bias

ATP-dependent and independent functions of Rad54 in genome maintenance

R ad54, a member of the SWI/SNF protein family of DNA-dependent ATPases, repairs DNA double-strand breaks (DSBs) through homologous recombination. Here we demonstrate that Rad54 is required for the timely accumulation of the homologous recombination proteins Rad51 and Brca2 at DSBs. Because replication protein A and Nbs1 accumulation is not affected by Rad54 depletion, Rad54 is downstream of DSB resection. Rad54-mediated Rad51 accumulation does not require Rad54's ATPase activity. Thus, our experiments demonstrate that SWI/SNF proteins may have functions independent of their ATPase activity. However, quantitative real-time analysis of Rad54 focus formation indicates that Rad54's ATPase activity is required for the disassociation of Rad54 from DNA and Rad54 turnover at DSBs. Although the non-DNA-bound fraction of Rad54 reversibly interacts with a focus, independent of its ATPase status, the DNA-bound fraction is immobilized in the absence of ATP hydrolysis by Rad54. Finally, we show that ATP hydrolysis by Rad54 is required for the redistribution of DSB repair sites within the nucleus

Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling

RNA-seq is increasingly used for quantitative profiling of small RNAs (for example, microRNAs, piRNAs and snoRNAs) in diverse sample types, including isolated cells, tissues and cell-free biofluids. The accuracy and reproducibility of the currently used small RNA-seq library preparation methods have not been systematically tested. Here we report results obtained by a consortium of nine labs that independently sequenced reference, 'ground truth' samples of synthetic small RNAs and human plasma-derived RNA. We assessed three commercially available library preparation methods that use adapters of defined sequence and six methods using adapters with degenerate bases. Both protocol- and sequence-specific biases were identified, including biases that reduced the ability of small RNA-seq to accurately measure adenosine-to-inosine editing in microRNAs. We found that these biases were mitigated by library preparation methods that incorporate adapters with degenerate bases. MicroRNA relative quantification between samples using small RNA-seq was accurate and reproducible across laboratories and methods