Comparative analysis of RNA sequencing methods for degraded or low-input samples

Adiconis, Xian; Borges-Rivera, Diego; Satija, Rahul; DeLuca, David S.; Busby, Michele; Berlin, Aaron M.; Sivachenko, Andrey; Thompson, Dawn; Wysoker, Alec; Fennell, Timothy; Gnirke, Andreas; Pochet, Nathalie; Regev, Aviv; Levin, Joshua Z.

doi:10.1038/nmeth.2483

Cited by 423 publications

(471 citation statements)

References 26 publications

(41 reference statements)

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“…40 Mapped reads were visualized on the UCSC Genome Browser. RNA-seq library quality was assessed as described by Adiconis et al 41 Basically, RNA-seq library quality in small samples is assessed in terms of the fraction of mapped reads versus total number of reads and the fraction of reads mapped to genomic features (in our case, exons). We used a threshold of 70% of total reads mapped to the reference genome (Supplemental Dataset 1).…”

Section: Rna-seqmentioning

confidence: 99%

Deep Sequencing in Microdissected Renal Tubules Identifies Nephron Segment–Specific Transcriptomes

Lee¹,

Chou²,

Knepper³

2015

Journal of the American Society of Nephrology

464

480

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The function of each renal tubule segment depends on the genes expressed therein. High-throughput methods used for global profiling of gene expression in unique cell types have shown low sensitivity and high false positivity, thereby limiting the usefulness of these methods in transcriptomic research. However, deep sequencing of RNA species (RNA-seq) achieves highly sensitive and quantitative transcriptomic profiling by sequencing RNAs in a massive, parallel manner. Here, we used RNA-seq coupled with classic renal tubule microdissection to comprehensively profile gene expression in each of 14 renal tubule segments from the proximal tubule through the inner medullary collecting duct of rat kidneys. Polyadenylated mRNAs were captured by oligo-dT primers and processed into adapter-ligated cDNA libraries that were sequenced using an Illumina platform. Transcriptomes were identified to a median depth of 8261 genes in microdissected renal tubule samples (105 replicates in total) and glomeruli (5 replicates). Manual microdissection allowed a high degree of sample purity, which was evidenced by the observed distributions of well established cell-specific markers. The main product of this work is an extensive database of gene expression along the nephron provided as a publicly accessible webpage (https://helixweb.nih.gov/ESBL/Database/NephronRNAseq/index.html). The data also provide genomewide maps of alternative exon usage and polyadenylation sites in the kidney. We illustrate the use of the data by profiling transcription factor expression along the renal tubule and mapping metabolic pathways.

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Section: Rna-seqmentioning

confidence: 99%

Deep Sequencing in Microdissected Renal Tubules Identifies Nephron Segment–Specific Transcriptomes

Lee¹,

Chou²,

Knepper³

2015

Journal of the American Society of Nephrology

464

480

View full text Add to dashboard Cite

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“…Transcriptome analysis of osteocyte gene expression from tumor-bearing bones relative to naive samples was performed following RNA extraction from homogenized (TriReagent, Sigma-Aldrich) osteocyte-enriched bone, quality checks by Total RNA Pico Chip (Agilent Technologies), and depletion of ribosomal RNA using RNaseH (Epicentre) and ribosomal RNA-targeting oligonucleotides. 46 Total-RNA library preparation was performed by using the TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina). Samples were sequenced on a HiSequation 2500 sequencer (Illumina).…”

Section: Osteocyte Rna-seq Analysismentioning

confidence: 99%

Inhibiting the osteocyte-specific protein sclerostin increases bone mass and fracture resistance in multiple myeloma

McDonald

Reagan

Youlten

et al. 2017

Blood

162

161

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Multiple myeloma (MM) is a plasma cell cancer that develops in the skeleton causing profound bone destruction and fractures. The bone disease is mediated by increased osteoclastic bone resorption and suppressed bone formation. Bisphosphonates used for treatment inhibit bone resorption and prevent bone loss but fail to influence bone formation and do not replace lost bone, so patients continue to fracture. Stimulating bone formation to increase bone mass and fracture resistance is a priority; however, targeting tumor-derived modulators of bone formation has had limited success. Sclerostin is an osteocyte-specific Wnt antagonist that inhibits bone formation. We hypothesized that inhibiting sclerostin would prevent development of bone disease and increase resistance to fracture in MM. Sclerostin was expressed in osteocytes from bones from naive and myeloma-bearing mice. In contrast, sclerostin was not expressed by plasma cells from 630 patients with myeloma or 54 myeloma cell lines. Mice injected with 5TGM1-eGFP, 5T2MM, or MM1.S myeloma cells demonstrated significant bone loss, which was associated with a decrease in fracture resistance in the vertebrae. Treatment with anti-sclerostin antibody increased osteoblast numbers and bone formation rate but did not inhibit bone resorption or reduce tumor burden. Treatment with anti-sclerostin antibody prevented myeloma-induced bone loss, reduced osteolytic bone lesions, and increased fracture resistance. Treatment with anti-sclerostin antibody and zoledronic acid combined increased bone mass and fracture resistance when compared with treatment with zoledronic acid alone. This study defines a therapeutic strategy superior to the current standard of care that will reduce fractures for patients with MM

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“…[22][23][24][25][26] Furthermore, RNA-Seq on FFPE tissue has only rarely been used to detect gene fusions. 6,23,27 The study of Sweeney and co-workers is of particular interest in the present context, as they also investigated sarcomas (three synovial sarcomas, three MLS, two Ewing sarcomas, and one clear cell sarcoma), used Illumina equipment and kits, and had results similar to ours; 23 all their cases showed the expected gene fusions, although some of the samples had to be re-analyzed at greater depth or after depletion of ribosomal RNA before obtaining positive results.…”

Section: Rna-seq and Fish On Ffpe Sections From Bfh C Walther Et Almentioning

confidence: 99%

Gene fusion detection in formalin-fixed paraffin-embedded benign fibrous histiocytomas using fluorescence in situ hybridization and RNA sequencing

Walther

Hofvander

Nilsson

et al. 2015

Laboratory Investigation

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Benign fibrous histiocytomas (FH) can be subdivided into several morphological and clinical subgroups. Recently, gene fusions involving either one of two protein kinase C genes (PRKCB and PRKCD) or the ALK gene were described in FH. We here wanted to evaluate the frequency of PRKCB and PRKCD gene fusions in FH. Using interphase fluorescence in situ hybridization on sections from formalin-fixed paraffin-embedded (FFPE) tumors, 36 cases could be analyzed. PRKCB or PRKCD rearrangements were seen in five tumors: 1/7 regular, 0/3 aneurysmal, 0/6 cellular, 2/7 epithelioid, 0/1 atypical, 2/10 deep, and 0/2 metastatic lesions. We also evaluated the status of the ALK gene in selected cases, finding rearrangements in 3/7 epithelioid and 0/1 atypical lesions. To assess the gene fusion status of FH further, deep sequencing of RNA (RNASeq) was performed on FFPE tissue from eight cases with unknown gene fusion status, as well as on two FH and six soft tissue sarcomas with known gene fusions; of the latter eight positive controls, the expected fusion transcript was found in all but one, while 2/8 FH with unknown genetic status showed fusion transcripts, including a novel KIRREL/PRKCA chimera. Thus, also a third member of the PRKC family is involved in FH tumorigenesis. We conclude that gene fusions involving PRKC genes occur in several morphological (regular, cellular, aneurysmal, epithelioid) and clinical (cutaneous, deep) subsets of FH, but they seem to account for only a minority of the cases. In epithelioid lesions, however, rearrangements of PRKC or ALK were seen, as mutually exclusive events, in the majority (5/7) of cases. Finally, the study also shows that RNA-Seq is a promising tool for identifying gene fusions in FFPE tissues. Benign fibrous histiocytoma (FH) can be subdivided into several morphological and clinical subgroups. Morphological variants include cellular, aneurysmal, epithelioid, and atypical types, and clinical manifestations range from benign tumors of the skin, deep soft tissues or skeleton to, rarely, metastasizing tumors. [1][2][3] We recently showed that both cutaneous and deep FH may carry specific gene fusions, in which a membereither PRKCB and PRKCD-of the gene family encoding protein kinase C (PRKC) is juxtaposed with a gene encoding a membrane-associated protein. 4 The pathogenetic mechanism was assumed to involve the uncoupling of the carboxy-terminal kinase domain of the PRKC protein from its regulatory domain, and its ectopic localization through fusion with the amino-terminal part of a membrane-associated protein. It has also recently been shown that some cases of epithelioid, and possibly also atypical, FH may harbor fusions activating the ALK protein. 5,6 The connection between ALK rearrangements and FH was further evaluated and strengthened by Doyle et al., showing that ALK fusions were restricted to the epithelioid subtype. 7 To study the frequency and distribution of PRKCB and PRKCD fusions in FH, we used interphase fluorescence in situ hybridization (FISH) on a series of tumors that...

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Comparative analysis of RNA sequencing methods for degraded or low-input samples

Cited by 423 publications

References 26 publications

Deep Sequencing in Microdissected Renal Tubules Identifies Nephron Segment–Specific Transcriptomes

Deep Sequencing in Microdissected Renal Tubules Identifies Nephron Segment–Specific Transcriptomes

Inhibiting the osteocyte-specific protein sclerostin increases bone mass and fracture resistance in multiple myeloma

Gene fusion detection in formalin-fixed paraffin-embedded benign fibrous histiocytomas using fluorescence in situ hybridization and RNA sequencing

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