The adequate planning of sequencing experiments so as to avoid technical biases is as important as good experimental design, especially when the experiment involves a large number of samples that need to be processed in several batches. In this case, including controls, randomizing sample processing and smart management of sequencing runs are crucial to obtain error-free data (Fig. 1a; Box 2).
When a reference genome is available, RNA-seq analysis will normally involve the mapping of the reads onto the reference genome or transcriptome to infer which transcripts are expressed. Mapping solely to the reference transcriptome of a known species precludes the discovery of new, unannotated transcripts and focuses the analysis on quantification alone. By contrast, if the organism does not have a sequenced genome, then the analysis path is first to assemble reads into longer contigs and then to treat these contigs as the expressed transcriptome to which reads are mapped back again for quantification. In either case, read coverage can be used to quantify transcript expression level (Fig. 1b). A basic choice is whether transcript identification and quantification are done sequentially or simultaneously.
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Read mapping and transcript identification strategies. Three basic strategies for regular RNA-seq analysis. a An annotated genome is available and reads are mapped to the genome with a gapped mapper. Next (novel) transcript discovery and quantification can proceed with or without an annotation file. Novel transcripts are then functionally annotated. b If no novel transcript discovery is needed, reads can be mapped to the reference transcriptome using an ungapped aligner. Transcript identification and quantification can occur simultaneously. c When no genome is available, reads need to be assembled first into contigs or transcripts. For quantification, reads are mapped back to the novel reference transcriptome and further analysis proceeds as in (b) followed by the functional annotation of the novel transcripts as in (a). Representative software that can be used at each analysis step are indicated in bold text. Abbreviations: GFF General Feature Format, GTF gene transfer format, RSEM RNA-Seq by Expectation Maximization
PacBio RNA-seq is the long-read approach with the most publications to date. The technology has proven useful for unraveling isoform diversity at complex loci [186], and for determining allele-specific expression from single reads [187]. Nevertheless, long-read sequencing has its own set of limitations, such as a still high error rate that limits de novo transcript identifications and forces the technology to leverage the reference genome [188]. Moreover, the relatively low throughput of SMRT cells hampers the quantification of transcript expression. These two limitations can be addressed by matching PacBio experiments with regular, short-read RNA-seq. The accurate and abundant Illumina reads can be used both to correct long-read sequencing errors and to quantify transcript levels [189]. Updates in PacBio chemistry are increasing sequencing lengths to produce reads with a sufficient number of passes over the cDNA molecule to autocorrect sequencing errors. This will eventually improve sequencing accuracy and allow for genome-free determination of isoform-resolved transcriptomes.
Conclusion: A holistic description of the developing immunome was obtained with key developmental milestones in the T cell compartment identified. This atlas has the translational potential of helping us define the stage of immune maturity and identify the pathological cell subset in both paediatric and adult immune mediated diseases by the direct comparison with this reference atlas using the web-based portal that will be made freely available.
Results: During 24 months 59% (19 (3 oligo)/31 (61%) of patients in arm 1, 16 (1 oligo)/32 (50%) in arm 2 and 19 (1 oligo)/29 (65%) in arm 3) had tapered and stopped all DMARDs (drug-free inactive disease) after median 15.0 (IQR 12.0-18.0) months (arm 1), 19.5 (12.0-24.0) months (arm 2) and 18.0 (12.0-21.0) months (arm 3) of therapy. However, 26% (6 (1 oligo) patients in arm 1, 3 in arm 2 and 5 in arm 3) subsequently had to restart treatment before end of study, after median time-to-flare 18.0 (15.0-21.0) months (p=0.7).
Conclusion: During 24 months of treatment to target inactive disease, including tapering and stop-strategies, flare frequency was 26% after median 18 months. After 24 months, 71% of patients had inactive disease and 39% were in drug-free inactive disease.
Three patients achieved drug-free remission. Median dose of prednisolone (PSL) was 0.8 (0.4-2.4) mg/kg/day at initiation of treatment and 0 (0-0.3) mg/kg/day at the last visit. Immunosuppressants were used in 13 (54%) from the onset and in 21 (88%) at the last visit. IVCY was used in 10 patients (42%) from the onset, and 4 after the relapse. Some biologics were used in 4 (17%) from the onset, and in 12 (50%) at the last visit.
Methods: A 6-year-old girl was referred with a 4-days history of vomiting and abdominal pain. Her physicial examination was normal except abdominal tenderness. Initial investigations showed Hb 14.4 g/dl, WBC 41.8 10 9/L, platalet count 443 10 9/L, C-reactive protein 50.6 mg/L, erythrocyte sedimentation rate 1mm/hr, albumin: 1.9 g/dL. In abdominal ultrasonograpy hydrops of gallbaldder was detected. Few days later purpuric rash on her legs was observed. She was diagnosed as HSP and prednisolone at a dose of 2 mg/kg/day was given to her. Despite steroid treatment, back and chest pain added to abdominal pain. Increased amylase of 344 U/L and lipase 294 U/L levels were detected at the seventh day of disease, but ultrasonographic pancreatitis was not observed. In addition to fat free diet intravenous fluids and pulse steroid treatment (30 mg/kg, for 3 days) was given. In the second week of the disease proteinüria was detected in 24 hour urine testing, and mesengioproliferative glomerulonephritis was diagnosed in renal biopsy. Pulse cyclophosphamide treatment was started her for HSP nephritis. Her pancreatitis resolved within a month
Methods: Using existing literature (including grey literature), an online survey (consisted of 152 questions, 29 items related to young adults two of which were free text questions) was developed and sent to National Rheumatoid Arthritis Society (NRAS) members and distributed to non-members via social media tools including Facebook, Twitter and HealthUnlocked. Data collected included views and experiences in career planning and employment. The data pertaining to young adults are presented here.
Introduction: Familial Mediterranean fever (FMF) is the most common autoinflammatory disease with autosomal recessive inheritance. There is MEFV gene mutation encoding the pyrine protein in the short arm of chromosome 16 that leads to overexpression of IL-1. The basic treatment in FMF has been colchicine since 1972. Colchicine is effective both in the prevention and treatment of attacks, and in reducing the frequency of amyloidosis. However, there is resistance to colchicine in 5-10% of FMF cases, and anti interleukin-1 (anti IL-1) is effective in these cases.
We describe an 18 year-old boy previously treated with several non-biologic DMARDS in combination with TNF-blockade, Il-1 blockade and IL-6 blockade. He has never been free of inflammation and always needed oral corticosteroids (0.15-0.20 mg/kg). His main clinical problems have been arthritis and uveitis. During the last 5 years he had intermittent headache that seems to correlate with flares of the disease. In December 2017 his headache became worse.
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