| 1. |
- Jemt, Anders, 1985-, et al.
(författare)
-
Evaluation of methods for whole genome and transcriptome sequencing from nanograms of FFPE samples
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The most widely used method for the preservation of clinical tissue specimens is formalin fixation and paraffin embedding (FFPE). Simultaneous analysis of RNA and DNA from samples preserved using this method have long proved problematic, primarily due to lack of material. Here, we describe an attempt to build a complete analysis package for RNA and DNA extracted from single tissue sections. The workflow includes quality control of the extracted material, library preparation and data analysis. We extract DNA with varying integrity from FFPE sections and subject them to whole genome sequencing using two library preparation methods, Illumina TruSeq Nano using the Illumina NeoPrep and Rubicon Genomics ThruPlex. We are able to obtain some usable data, albeit with high duplication rates, demonstrating both the possibilities and challenges of sequencing damaged DNA. Two different approaches to transcriptome sequencing are assessed, the TruSeq RNA Access library preparation kit from Illumina and the SMARTer Stranded Total RNA-Seq Kit - Pico Input from Clonetech. The sequence capture approach of the TruSeq kit is shown to be more robust to low integrity RNA compared to the SMARTer kit. However, the SMARTer kit needs much less starting material and is able to yield data about all transcripts, not just protein coding mRNA.
|
|
| 2. |
- Jemt, Erik, et al.
(författare)
-
Outcomes in Emergency Department Patients with Dyspnea versus Chest Pain : A Retrospective Consecutive Cohort Study
- 2022
-
Ingår i: Emergency Medicine International. - : Hindawi Limited. - 2090-2840 .- 2090-2859. ; 2022, s. 1-7
-
Tidskriftsartikel (refereegranskat)abstract
- Dyspnea and chest pain are major and important causes of contact at the emergency department (ED). Dyspnea is associated with high morbidity and mortality, but data on characteristics and outcomes compared with chest pain in the ED are limited. This was a retrospective cohort study of consecutive patients with contact causes of dyspnea or chest pain at two Swedish EDs from 2010 to 2014. Hospital admittance, ED revisits, and mortality were analyzed using multivariable regression models, adjusted for ED and markers of disease severity (age, sex, centre, Charlson comorbidity index, c-reactive protein, troponin T, and arrival by ambulance). 29,291 patients (mean age 58.3 years; 48.9% women) with dyspnea (n = 8,812) or chest pain (n = 20,479) were included. Dyspnea patients were older than patients with chest pain (64 vs. 56 years, p < 0.001) and had more comorbidity and higher average blood troponin T and c-reactive protein levels. Dyspnea patients also had higher hospitalization rates (48% vs. 30%; adjOR (95% CI) 2.1-2.3), including the intensive care unit (1.4% vs. 0.1%; adjOR 6.9-15.9), and more ED revisits (11% vs. 7%; adjOR 1.2-1.7) in 30 days. Dyspnea patients had five-fold increased mortality compared to those with chest pain; hazard ratio (HR) 5.1 (4.8-5.4), adjusted for markers of disease severity, the mortality was two-fold higher, HR 2.2 (2.0-2.4). Compared with chest pain patients, ED dyspnea patients are older, have more comorbidity, and have worse outcomes in terms of hospitalization, morbidity, and mortality.
|
|
| 3. |
- Lundin, Sverker, et al.
(författare)
-
Endonuclease specificity and sequence dependence of Type IIS restriction enzymes
- 2015
-
Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 10:1
-
Tidskriftsartikel (refereegranskat)abstract
- Restriction enzymes that recognize specific sequences but cleave unknown sequence outside the recognition site are extensively utilized tools in molecular biology. Despite this, systematic functional categorization of cleavage performance has largely been lacking. We established a simple and automatable model system to assay cleavage distance variation (termed slippage) and the sequence dependence thereof. We coupled this to massively parallel sequencing in order to provide sensitive and accurate measurement. With this system 14 enzymes were assayed (AcuI, BbvI, BpmI, BpuEI, BseRI, BsgI, Eco57I, Eco57MI, EcoP15I, FauI, FokI, GsuI, MmeI and SmuI). We report significant variation of slippage ranging from 1-54%, variations in sequence context dependence, as well as variation between isoschizomers. We believe this largely overlooked property of enzymes with shifted cleavage would benefit from further large scale classification and engineering efforts seeking to improve performance. The gained insights of in-vitro performance may also aid the in-vivo understanding of these enzymes.
|
|
