Takaho A. Endo. Quality control method for RNA-seq using single nucleotide polymorphism allele frequency. Genes to Cells Published online: 21 SEP 2014. DOI: 10.1111/gtc.12178
RNA sequencing (RNA-seq) provides information not only about the level of expression of individual genes but also about genomic sequences of host cells. When we use transcriptome data with whole-genome single nucleotide polymorphism (SNP) variant information, the allele frequency can show the genetic composition of the cell population and/or chromosomal aberrations. Here, I show how SNPs in mRNAs can be used to evaluate RNA-seq experiments by focusing on RNA-seq data based on a recently retracted paper on stimulus-triggered acquisition of pluripotency (STAP) cells. The analysis indicated that different types of cells and chromosomal abnormalities might have been erroneously included in the dataset. This re-evaluation showed that observing allele frequencies could help in assessing the quality of samples during a study and with retrospective evaluation of experimental quality.
(http://onlinelibrary.wiley.com/doi/10.1111/gtc.12178/abstract)
科研費に採択されるための最良の方法は,「書き上げた申請書を誰かに見せて添削してもらうこと」だと思う.見てもらう人が採択経験豊富な人ならなおよい.そういった人に申請書を見てもらって何度も何度も直すのが一番よい方法だ.(小噺その10:科研費に採択されるための最良の方法 Smart Lab Life 羊土社)
サイエンスへ投稿されたときの査読内容(全文へのリンク retractionwatch.comウェブサイト内)
論文タイトル:Stress altered somatic cells capable of forming an embryo
時期:2012年8月21日
Reviewer 1: ” … This is such an extraordinary claim that a very high level of proof is required to sustain it and I do not think this level has been reached. I suspect that the results are artifacts derived from the following processes: (1) the tendency of cells with GFP reporters to go green as they are dying. (2) the ease of cross contamination of cell lines kept in the same lab. …”
Reviewer 2: ” … Unfortunately, the paper presents only a superficial description of many critical aspects of the work. …”
Reviewer 3: “… If these results are repeatable, a paradigm of developmental biology would be changed. …”
”The DNA analysis of the chimeric mice is the only piece of data that does not fit with the contamination theory. But the DNA fragments in the chimeras don’t look the same as those in the lymphocytes. This assay is not properly explained. If it is just an agarose gel then the small bands could be anything. Moreover this figure has been reconstructed. It is normal practice to insert thin white lines between lanes taken from different gels (lanes 3 and 6 are spliced in). Also I find the leading edge of the GL band suspiciously sharp in #2-#5.”
Referee #1: ” … This is a very interesting manuscript and potentially groundbraking. However, the presentation and data supporting the conclusions are somewhat speculative and, in some cases, preliminary. …”
Refree #2: ” … Most convincing, however, would be to demonstrate visually by time lapse tracking of single cells conversion of CD45 immunofluorescent cells into CD45 negative/Oct4 GFP positive cells that can also be stained with Ecadherin and/or Nanog. …”
Refree #3: “… the claim that these cells are pluripotent is not fully validated. The possibility remains that the tissues formed in vitro or in teratomas or chimeras are derived from multiple (perhaps partially ) reprogrammed cells, each with limited differetiation capacity. The authors might take advantage of the fact that some of the reprogrammed cells have T-cell receptor rearrangements to elucidate whether the differentiated cells in teratomas or mice are clonally derived. …”
NATUREに同時に投稿されたレター論文「Developmental potential for embryonic and placental lineages in reprogrammed cells with acquired pluripotency」に対する査読者のコメント(2013年4月4日)
Referee #1: ” .. it is important to realize not single experimetn in any of the two manuscript evaluates the “quality” of the cells, performs comparative genome-wide analysis or precise quantifiable assays side-by-side with ESCs/iPSCs. ..”
Referee #2: “. .. they do not decisively illuminate the identity of STAP cells. …”
Referee #3: ” … It is important to report the properties of clonally derived STAP ES like stem cells, otherwise, it is not clear whter one cell population gives rise to all the lineages in teratomas or in chimeras. …”
“While they find your work of great potential interest, as do we, they have raised important concerns that in our view need to be addressed before we can consider publication in Nautre. Should further experimental data allow you to address these criticisms, we would be happy to look at a revised manuscript (unless something similar has been accepted at Nature or appeared elsewhere in the meantime). …””
この後、2回の改訂を経て最終的にネイチャーに受理されたようです。
Obokata et al., Stimulus-triggered fate conversion of somatic cells into pluripotency. Nature 505, 641–647 (30 January 2014)
Obokata et al., Bidirectional developmental potential in reprogrammed cells with acquired pluripotency. Nature 505, 676–680 (30 January 2014)
Received 10 March 2013
Accepted 20 December 2013
Published online 29 January 2014
Retraction (July, 2014)
“.. Yoshiki struck me as a happy scientist. He spoke softly and with a unique smile as he described Japanese traditions or revealed his astonishing findings. .. Sasai was a master at deciphering the code by which cells learn their place in a developing embryo. ..Yoshiki Sasai (1962–2014):Stem-cell biologist who decoded signals in embryos. Arturo Alvarez-Buylla. Nature 513,34 (04 September 2014) doi:10.1038/513034a
“.. Yoshiki had an unmatched ability to decipher the embryo—specifically, to uncover how this developmental marvel generates the extraordinary diversity of cell types that become organized into unique structures, like the pituitary gland, the brain, or the eye. .. “Obituary Yoshiki Sasai (1962–2014). Arnold R. Kriegsteinemail DOI: http://dx.doi.org/10.1016/j.stem.2014.08.007 Cell Stem Cell Volume 15, Issue 3, p265–266, 4 September 2014
“.. Yoshiki had a unique ability to see things clearly while others were left wandering in the dark. .. “OBITUARY Yoshiki Sasai: stem cell Sensei
Stefano Piccolo Development (2014) 141, 1-2 doi:10.1242/dev.116509