Response to “The Reality of Pervasive Transcription” 英文参考文献.docVIP

Perspective Responseto‘‘TheRealityofPervasiveTranscription’’ HarmvanBakel1,CoreyNislow1,2,BenjaminJ.Blencowe1,2,TimothyR.Hughes1,2 * 1Banting and Best Department of Medical Research and Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada,2DepartmentofMolecularGenetics,UniversityofToronto,Toronto,Ontario,Canada Clark et al. criticize several aspects of our study [1], and specifically challenge our assertion that the degree of pervasive transcriptionhaspreviouslybeenoverstat- ed. We disagree with much of their reasoning and their interpretation of our work. For example, many of our conclu- sions are based on overall sequence read distributions, while Clark et al. focus on tic’’ transcripts greatly increases their abundance[7,8]. emphasizedthelackofabundantpervasive transcriptioninourstudy.Clarketal.cite papers that have previously documented pervasivetranscription,andpointoutthat several different approaches have been usedasconfirmation.WebelievethatClark et al. misinterpret what can be claimed frommuchoftheliteratureinthisarea,and fail to acknowledge known weaknesses in some of these studies. We previously reviewed these issues [9]. For example, the number of transfrags detected in permutedtilingarraydatacanbeashigh asitisintherealdata[10].Inaddition,a common form of ‘‘validation’’ in these papers is RT-PCR or RACE, but these approachesaregenerallysemi-quantitative at best and are prone to artefacts such as templateswitching,whichreadilyproduces chimeric transcripts in vitro ([11] and references therein). Indeed, we note that intheENCODE1%study[2]repeatedly citedbyClarketal.,75ofthe100negative controls (randomly selected non-transfrag regions)wereactuallydetectedbyRACE, making the ‘‘validation’’ rate for negative controlsonlyslightlylowerthanthatforthe intronic and intergenic transfrags (86%– 88%). Thus, either the tiling arrays or RACEassaysarehighlyerrorprone.The contentionofClarketal.that‘‘anyesti