13 research outputs found
Energy spread of ultracold electron bunches extracted from a laser cooled gas
Ultrashort and ultracold electron bunches created by near-threshold
femtosecond photoionization of a laser-cooled gas hold great promise for
single-shot ultrafast diffraction experiments. In previous publications the
transverse beam quality and the bunch length have been determined. Here the
longitudinal energy spread of the generated bunches is measured for the first
time, using a specially developed Wien filter. The Wien filter has been
calibrated by determining the average deflection of the electron bunch as a
function of magnetic field. The measured relative energy spread
agrees well with the theoretical model
which states that it is governed by the width of the ionization laser and the
acceleration length
Evaluation of protocols for rRNA depletion-based RNA sequencing of nanogram inputs of mammalian total RNA.
Next generation RNA-sequencing (RNA-seq) is a flexible approach that can be applied to a range of applications including global quantification of transcript expression, the characterization of RNA structure such as splicing patterns and profiling of expressed mutations. Many RNA-seq protocols require up to microgram levels of total RNA input amounts to generate high quality data, and thus remain impractical for the limited starting material amounts typically obtained from rare cell populations, such as those from early developmental stages or from laser micro-dissected clinical samples. Here, we present an assessment of the contemporary ribosomal RNA depletion-based protocols, and identify those that are suitable for inputs as low as 1-10 ng of intact total RNA and 100-500 ng of partially degraded RNA from formalin-fixed paraffin-embedded tissues
Increasing quality, throughput and speed of sample preparation for strand-specific messenger RNA sequencing
Background
RNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. However, standard approaches for library construction typically require relatively high amounts of input RNA, are labor intensive, and are time consuming.
Methods
Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction.
Results
This work has resulted in the identification of an optimized messenger RNA isolation protocol, a potent reverse transcriptase for cDNA synthesis, and an efficient chemistry and a simplified formulation of library construction reagents. We also present an optimization of bead-based purification and size selection designed to maximize the recovery of cDNA fragments.
Conclusions
These developments have allowed us to assemble a rapid high throughput pipeline that produces high quality data from amounts of total RNA as low as 25Â ng. While the focus of this study is on RNA-seq sample preparation, some of these developments are also relevant to other next-generation sequencing library types.Medicine, Faculty ofScience, Faculty ofNon UBCMedical Genetics, Department ofMicrobiology and Immunology, Department ofReviewedFacult
P658: Detection of variant parent-of-origin in diverse hereditary cancer syndromes using only the proband’s blood sample
Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples
Automated high throughput nucleic acid purification from formalin-fixed paraffin-embedded tissue samples for next generation sequence analysis
<div><p>Curation and storage of formalin-fixed, paraffin-embedded (FFPE) samples are standard procedures in hospital pathology laboratories around the world. Many thousands of such samples exist and could be used for next generation sequencing analysis. Retrospective analyses of such samples are important for identifying molecular correlates of carcinogenesis, treatment history and disease outcomes. Two major hurdles in using FFPE material for sequencing are the damaged nature of the nucleic acids and the labor-intensive nature of nucleic acid purification. These limitations and a number of other issues that span multiple steps from nucleic acid purification to library construction are addressed here. We optimized and automated a 96-well magnetic bead-based extraction protocol that can be scaled to large cohorts and is compatible with automation. Using sets of 32 and 91 individual FFPE samples respectively, we generated libraries from 100 ng of total RNA and DNA starting amounts with 95–100% success rate. The use of the resulting RNA in micro-RNA sequencing was also demonstrated. In addition to offering the potential of scalability and rapid throughput, the yield obtained with lower input requirements makes these methods applicable to clinical samples where tissue abundance is limiting.</p></div
Automated high throughput FormaPure-based extraction protocol.
<p>(<b>A</b>) Work flow illustration of sample acquisition, upstream sample processing and extraction. Note that a separate high temperature incubation step is added to facilitate the reversal of remaining crosslinks. The upstream processes are manual in the original protocol whereas those steps are modified to be suitable for automation in the modified protocol. The in-house on-deck heating blocks were instrumental in rendering the lysis/deparaffinization steps automatable. Acquisition of samples in SBS format matrix tubes with their automated capping and decapping were also further measures that allowed the entire process to be amenable for automated liquid handling. (<b>B</b>) gDNA yield. Historical gDNA yield data from the Qiagen/High Pure protocol (Q; n = 142) using equivalent sizes of numerous FFPE samples of lymphoma origin was compared with that of the FormaPure protocol (F; n-91). (C) RNA yield. Comparison of the Qiagen-High Pure (Q-H), and FormaPure (F) protocols are shown. N = 142 for Q-H and N = 44 for F.</p
Suitability of the FormaPure extracted RNA for FFPE strand-specific RNA-seq.
<p>(<b>A)</b> Strand-specific libraries were generated from four different FormaPure extracted human FFPE samples (FFPE A-D) and UHR fresh RNA. Two different total (DNase-treated) RNA input amounts were used (100 and 200 ng, respectively). Final library yield (nM) (left panel) and % duplicates (middle panel) as well as the distribution of aligned reads to various regions of the transcriptome (right panel) are shown graphically. These libraries were sequenced as a pool at PE75 bp. (<b>B</b>) Comparison of Qiagen and FormaPure extraction protocols using mouse FFPE scrolls. Final library yield (nM) (Left panel) and % duplicates, % aligned, and the distribution of aligned reads to various regions of the transcriptome (middle panel) as well as number of genes with 1x coverage (right panel) are shown graphically.</p