ModENCODE	  RNA-­‐Seq	  best	  practices	  (DRAFT)	  adapted	  from	  Myers/Wold/Gingeras	  2009	  	  Send	  any	  comments	  on	  this	  document	  to	  Brian	  Oliver	  	  1.	  Obtain	  greater	  than	  1X	  RNA-­‐Seq	  coverage	  of	  known	  "gold	  standard"	  transcripts	  that	  are	  present	  in	  a	  range	  of	  abundances.	  For	  cell	  lines	  this	  should	  include	  transcripts	  present	  at	  less	  than	  1	  copy	  per	  cell.	  
This	  standard	  will	  be	  difficult	  to	  achieve	  in	  complex	  tissues	  where	  rare	  transcripts	  are	  expressed	  in	  rare	  cell	  types.	  Cell	  level	  purification,	  fractionation,	  normalization	  or	  other	  methods	  for	  getting	  rare	  transcripts	  from	  organisms	  will	  be	  required.	  	  2.	  Primary	  determination	  will	  be	  performed	  in	  biological	  duplicate,	  unless	  there	  is	  a	  compelling	  reason	  indicating	  that	  this	  is	  impractical	  or	  wasteful	  (e.g.	  overlapping	  timepoints,	  Solexa	  &	  454	  data).	  Technical	  replicates	  of	  the	  same	  library	  are	  not	  required.	  Biological	  duplicate	  must	  show	  >	  0.9	  correlation	  for	  transcripts/	  features	  greater	  than	  1	  RPKM.	  	  3.	  A	  ladder	  of	  RNA	  spike-­‐ins	  should	  be	  included	  to	  calibrate	  quatification,	  sensitivity,	  coverage	  and	  linearity.	  The	  pools	  of	  ERCC	  mRNAs	  obtained	  from	  NIST	  are	  available	  and	  should	  be	  used	  in	  future	  experiments.	  	  4.	  A	  null	  model	  for	  non-­‐transcribed	  regions	  is	  highly	  problematic	  as	  we	  do	  not	  know	  enough	  about	  the	  genome	  and	  how	  much	  of	  it	  is	  really	  transcribed.	  While	  we	  do	  not	  have	  a	  model	  for	  biological	  transcriptional	  noise,	  we	  should	  determine	  the	  frequency	  of	  sequencing	  and	  mis-­‐mapping	  in	  simulations	  to	  determine	  how	  much	  density	  and	  junction	  noise	  is	  expected	  for	  technical	  reasons.	  	  5.	  Read	  density	  provides	  strong	  support	  for	  transcription	  of	  a	  given	  segment	  of	  the	  genome,	  but	  producing	  a	  full	  annotation	  of	  all	  spliced	  transcripts	  is	  complicated	  by	  read	  length	  and	  strandedness.	  
Longer	  and	  longer	  RNA-­‐seq	  read	  lengths	  are	  possible	  and	  maximum	  length	  paired-­‐end	  data	  is	  highly	  desirable	  for	  determining	  junctions	  linking	  one	  or	  more	  exons.	  Overlapping	  genes	  should	  be	  distinguished	  by	  data	  from	  stranded	  libraries.	  Maximal	  discovery	  and	  annotation	  may	  require	  a	  mix	  of	  RNA-­‐Seq	  strategies.	  	  RNA-­‐seq	  best	  practices	  page	  2009	  1	  6.	  RNA-­‐Seq	  data	  currently	  provides	  very	  high	  quality	  exon-­‐pair	  or	  "genelet"	  data,	  but	  deduced	  full-­‐length	  transcript	  models	  produced	  exclusively	  by	  RNA-­‐Seq	  data	  are	  suspect.	  Exon-­‐pair/genelet	  annotation	  should	  be	  reported.	  Full-­‐length	  sequence	  should	  be	  pursued	  whenever	  feasible	  to	  provide	  connectivity.	  	  7.	  These	  guidelines	  will	  continue	  to	  evolve	  as	  the	  technology	  improves.	  

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