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Troubleshooting – Common Amplification and CE Errors/ Issues
 

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Troubleshooting – Common Amplification and CE Errors/ Issues presentation given at the 2013 Life Technologies Forensic Seminar Series

Troubleshooting – Common Amplification and CE Errors/ Issues presentation given at the 2013 Life Technologies Forensic Seminar Series

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    Troubleshooting – Common Amplification and CE Errors/ Issues Troubleshooting – Common Amplification and CE Errors/ Issues Presentation Transcript

    • Troubleshoo*ng  –  Common   Amplifica*on  and  CE  Errors/ Issues     April  Orbison   Sr.  HID  Field  Applica6ons  Specialist          
    • 9/18/13 | © Life Technologies™ 2 Overview   § Trouble  Shoo6ng  Tools   § Common  Capillary  Electrophoresis  Instrument   Issues  and  Observa6ons   § Common  Amplifica6on  Observa6ons    
    • 9/18/13 | © Life Technologies™ 3 Overview   § Trouble  Shoo6ng  Tools   § Common  Capillary  Electrophoresis  Instrument   Issues  and  Observa6ons   § Common  Amplifica6on  Observa6ons    
    • 9/18/13 | © Life Technologies™ 4 Troubleshoo*ng  Tools     Data  is  usually  viewed  in  the  following  order:   §  Raw  data     §  EPT  data/Status  view  (during  run)   §  Capillary  view   §  Instrument  logs   §  Analyzed  data   −  usually  seen  first  by  customer   §  Service  tools  and  soKware  (Field  Service  Engineer)      
    • 9/18/13 | © Life Technologies™ 5 Why  Review  Raw  Data  First?    Data  has  not  been  manipulated/analyzed  by   GeneMapper®  soKware   − No  baselining/No  smoothing   − No  peak  detec6on   − No  sizing   − No  allele  detec6on     − No  table  construc6on        
    • 9/18/13 | © Life Technologies™ 6 What  Raw  Data  Should  Look  Like!    
    • 9/18/13 | © Life Technologies™ 7 Poor  Raw  Data   Data  analyzed  correctly,  but  precursor  to  a  problem    
    • 9/18/13 | © Life Technologies™ 8 Poor  Raw  Data  
    • 9/18/13 | © Life Technologies™ 9 Review  Raw  Data:  Internal  Size  Standard   §  If  prepared  properly,  the  size  standard  is  incorporated  into   every  sample  along  with  HI-­‐DITM  Formamide   §  Scan  for  paSern,  resolu6on,  peak  morphology  and  peak   heights  of  the  size  standard     −  Can  be  used  to  determine  if  the  instrument  hardware  and  consumables   (i.e.  polymer,  buffer,  capillary/array)  are  working  properly   −  In  other  words,  can  help  you  determine  if  the  problem  is  related  to   capillary  electrophoresis   §  Just  takes  a  quick  review  of  the  raw  data  for  EACH  sample  
    • 9/18/13 | © Life Technologies™ 10 Troubleshoo*ng  Tool:  GS500  LIZ  &  GS500  ROX   §  All  present   §  Good  morphology   §  Clean  baseline   §  Adequate  signal  intensity   §  Rela6vely  balanced  peak  heights   §  Check  migra6on  by  reviewing  250  bp  peak  (analyzed  data)   GeneScan™ 500 LIZ® Internal Lane Size Standard triplet 75 1st doublet 450
    • 9/18/13 | © Life Technologies™ 11 Troubleshoo*ng  Tool:  GS600  LIZ®   Size  Standard  
    • 9/18/13 | © Life Technologies™ 12 Review  Raw  Data:  Evaluate  Ladders   §  Ensure  size  standard  and  ladder  peaks  are  present  and  have  good   morphology   §  Ensure  ladder  peak  heights  are  at  least  above  Peak  Amplitude  Threshold   (PAT)   §  Must  have  at  least  one  passing  ladder  per  plate   −  If  there  is  only  one  ladder  and  it  fails,  en6re  plate  must  be  rerun   §  If  you  are  using  GMID  3.X,  remove  any  failing  ladders  prior  to  analysis   −  If  not  removed,  they  will  all  get  averaged…throws  off  good  ladders   −  GMIDX  soKware  will  automa6cally  ignore  failing  ladders  
    • 9/18/13 | © Life Technologies™ 13 Review  Raw  Data:  Evaluate  Ladders  
    • 9/18/13 | © Life Technologies™ 14 Use  of  the  Size  Standard  &  Allelic  Ladder   Good Quality Size Standard/Ladder Profile Poor Quality Size Standard/ Ladder Profile Good Quality Result from Unknown DNA Sample(s) Single Sample or Few Samples: All Samples: Poor Quality Result from Unknown DNA Sample(s) Single Sample or Few Samples: All Samples: No Issues Investigate sample history and all steps prior to electrophoresis (extraction method, quant results, amplification parameters) Verify master mix made properly (master mix dispensed in well, insufficient mixing) Investigate size standard (expiration, storage) & ladder Possibly bubbles/poor injections Investigate instrument, reagents & consumables
    • 9/18/13 | © Life Technologies™ 15 Review  Raw  Data:  Check  Peak   Heights  &  Analysis  Range   Analysis Start Point -past primer peak -before 75 peak Analysis Stop Point Anywhere after 450 peak**
    • 9/18/13 | © Life Technologies™ 16 EPT  Data/Status  View   §  Compare  a  known,  good  EPT  file  with  problem  file     §  Ambient  temperature   §  Oven  temperature     §  Be  aware  that  default  run  voltage,  injec6on  voltage,  laser   power,  dynamic  range  and  injec6on  6me  vary  between  CE   pla_orms   EPT = Electrophoresis, Power & Temperature
    • 9/18/13 | © Life Technologies™ 17 Default  Run  Parameters     §  3130  and  3130xl   −  Run  voltage:  15,000   −  Injec6on  voltage:  3,000   −  Laser  Power:  15   −  Injec6on  Time:  3130  =  5  sec      3130xl  =  10  sec   −  Typical  camera  satura6on  =    ~7,000+     §  3500  and  3500xl   −  Run  voltage:  15,000    (13,000  for  Globalfiler  kits)   −  Injec6on  voltage:  1,200   −  Laser  Power:  10   −  Injec6on  Time:  3500  =  15  sec      3500xl  =  24  sec   −  Typical  camera  satura6on  =    ~24,000+     §  3730   −  Run    voltage:  15,000       −  Injec6on  voltage:  2,000   −  Laser  Power:  25   −  Injec6on  Time:  3730  =  10   −  Typical  camera  satura6on  =    ~24,000  +      
    • 9/18/13 | © Life Technologies™ 18 Naviga*ng  an  EPT  File     2 3 4 5 1 1.  Oven  &  cell  heater  stabilize   at  set  temperature   2.  Pre-­‐run     ●  equilibrates  ion  balance  in   system   3.  Injec6on     4.  Ini6al  separa6on     ●  voltage  stepped  up   gradually   5.  Separa6on   ●  data  collec6on  
    • 9/18/13 | © Life Technologies™ 19 EPT  Data    
    • 9/18/13 | © Life Technologies™ 20 Capillary  View:  What  to  Look  For   1.  Signal intensity 2.  Loss of resolution 3.  Migration issues 4.  Missing capillaries 5.  Possible patterns
    • 9/18/13 | © Life Technologies™ 21 Instrument  Logs   §  Can  tell  you   where  or   what  the   problem  is       §  Trust  them!  
    • 9/18/13 | © Life Technologies™ 22 Analyzed  Data       §  Analyzed  data  is  usually  what  people  oKen  view  first  and  deem  as  being   good  or  bad   §  This  spurs  ques6ons  and  concerns  and  may  develop  into  a  call  to  AB           §  Flip  side  is  data  may  seem  to  analyze  correctly,  yet  s6ll  have  problems   §  This  is  why  we  start  by  looking  at  the  Raw  Data  first    
    • 9/18/13 | © Life Technologies™ 23 Check  the  Controls:  AmpFℓSTR®  Kit   Posi*ve  Control   §  Sample  of  known  origin  that  is  provided  in  each  AmpFℓSTR®  kit  at  a  certain   concentra6on*   −  Different  kits  have  different  posi6ve  controls  that  may  be  supplied  at   different  concentra6ons   §  When  used  according  to  protocol  and  when  system  is  working  normally,   posi6ve  control  should  consistently  and  reliably  produce  the  same  results   each  and  every  6me   §  Primary  purpose  is  to  serve  as  a  genotyping  control  to  show  that   amplifica6on  progressed  normally   −  Can  also  help  diagnose  capillary  electrophoresis  problems  and  soKware   analysis  issues     *Posi6ve  controls  are  genotyping  controls  only,  not  quan6ta6on  controls      
    • 9/18/13 | © Life Technologies™ 24 Use  of  the  Posi*ve  Control   Good Quality Positive Control Result Poor Quality Positive Control Result Good Quality Result from Unknown DNA Sample(s) Single Batch: All Batches: Poor Quality Result from Unknown DNA Sample(s) Single Batch: All Batches: No Issues Investigate sample history, extraction method, quant results, dilution ratios, TE-4, sample inhibition/degradation Failure to add control DNA; See if reproducible Investigate control DNA (expiration, storage, contamination, etc.) Mistake made during master mix prep; TC failure; See if reproducible Investigate kit reagents (expiration, storage, contamination), TC (parameters, malfunction, temp verification)
    • 9/18/13 | © Life Technologies™ 25 Check  the  Controls:  Internal   Laboratory  QC  Controls   §  Known  standard  with  a  known  DNA  profile  from  the  customer's   lab  or  purchased  from  a  manufacturer     §  OKen  taken  through  the  en6re  process  from  extrac6on  through   data  analysis   §  Used  in  conjunc6on  with  the  previously  men6oned  controls  to   determine  if  the  en6re  process  is  working  properly   −  Can  help  determine  which  stage  of  the  process  had  an  error  
    • 9/18/13 | © Life Technologies™ 26 Overview   § Trouble  Shoo6ng  Tools   § Common  Capillary  Electrophoresis  Instrument   Issues  and  Observa6ons   § Common  Amplifica6on  Observa6ons    
    • 9/18/13 | © Life Technologies™ 27 Common  Capillary  Electrophoresis   Instrument  Issues  and  Observa*ons     § Front  –End  Troubleshoo6ng   § Migra6on  Problems   § Avoiding  Bubbles   § Shadow  Peaks  
    • 9/18/13 | © Life Technologies™ 28 Common  Capillary  Electrophoresis   Instrument  Issues  and  Observa*ons     § Front  –End  Troubleshoo6ng   § Migra6on  Problems   § Avoiding  Bubbles   § Shadow  Peaks  
    • 9/18/13 | © Life Technologies™ 29 Front-­‐End  Troubleshoo*ng   §  Common  Front-­‐End  Problems   −  Fluorescent  contamina6on   −  Low  or  no  signal   −  Failed  injec6ons   −  Loss  of  resolu6on   −  Inconsistent  peak  migra6on   −  Erra6c  EP  current   −  Extraneous  peaks  (spikes)  
    • 9/18/13 | © Life Technologies™ 30 What  is  the  Front-­‐End?   §  Pump  &  polymer  blocks   §  Buffer  &  water  reservoirs/jars   §  Ferrules  and  tubing   §  Capillary  array   §  Polymer,  buffer,  water   §  96-­‐well  plates  &  septa   The Front-End includes all replaceable hardware, reagents & consumables
    • 9/18/13 | © Life Technologies™ 31 Indica*ons  of  Possible  Front-­‐End   Problems     §  Allelic  ladder  and  size  standards  exhibit  the  same  problems  as  the   sample  data   §  Poor  data  occurs  across  mul6ple  plates   §  The  same  samples  run  on  another  instrument  do  not  exhibit  the   same  problems  
    • 9/18/13 | © Life Technologies™ 32 Fluorescent  Contamina*on   Possible  Causes   §  Poor  quality  water  or  reagents  (buffer,  polymer,  formamide)   §  Incorrect  or  infrequent  cleaning  of  system  components   §  Contamina6on  origina6ng  from  the  sample   §  Improper  use  of  canned  air  
    • 9/18/13 | © Life Technologies™ 33 Separated  Baseline  Due  to   Fluorescent  Contaminant  
    • 9/18/13 | © Life Technologies™ 34 No  Data  /  No  Signal   Possible  Causes   §  Injec*on  problem   −  Air  bubble  in  sample  tube   −  No  current  between  electrode  and   capillary  (air  bubble  in   electrophoresis  system)     −  Sample  volume  in  tubes  too  low   −  Sample  (&  size  standard)  not   added   −  Subop6mal  autosampler   calibra6on   −  Clogged/poor  capillary/array   §  Detec*on  problem   −  Dead  laser  (no  signal)   −  Poor  spa6al  calibra6on   Raw Data
    • 9/18/13 | © Life Technologies™ 35 Low  Signal   Possible  Causes   §  Poor  quality  system  reagents  (polymer,  formamide,  buffer,  water)   §  Incorrect  sample  prepara6on   §  Subop6mal  autosampler  calibra6on   §  Poor/improperly  stored/exhausted  array  or  capillary   §  Bubbles/Failure  to  centrifuge  plates   §  Contaminant  
    • 9/18/13 | © Life Technologies™ 36 Loss  of  Resolu*on  
    • 9/18/13 | © Life Technologies™ 37 Loss  of  Resolu*on   Possible  Causes   §  Poor  water  quality     §  Poor  quality  system  reagents   §  Insufficient  capillary  filling   −  Leak  in  the  system  fitngs   §  Air  in  the  system   −  Bubbles   §  Impuri6es   −  Protein,  salts   −  Detergents   §  Poor/exhausted  array   §  Poor  instrument  maintenance   Raw Data Analyzed Data
    • 9/18/13 | © Life Technologies™ 38 Raw Spikes:  Analyzed  vs.  Raw  Data   Analyzed
    • 9/18/13 | © Life Technologies™ 39 Example  of  Spikes  in  the  Allelic   Ladder  
    • 9/18/13 | © Life Technologies™ 40 Spikes:  Possible  Causes   Possible  Causes   §  Dust  or  lint  from  non-­‐lint  free   6ssues   §  Dried  polymer  deposits   §  Dried  buffer  deposits   §  Old  or  poor  quality  formamide   §  Air  bubbles   §  Electrical  surges   §  Poor/exhausted  capillary/array   §  Improper  use  of  canned  air     §  Use  of  powdered  gloves  
    • 9/18/13 | © Life Technologies™ 41 No*ce  a  Trend?   §  All  of  the  previously  men6oned  problems  share  many  of  the   same  causes   §  The  trend  will  con6nue…  
    • 9/18/13 | © Life Technologies™ 42 Steps  to  Resolve  Front-­‐End  Problems     §  Check  array  for  damage  (detec6on  cell  and  load  end)   §  Perform  water  wash  using  high  quality  water  source   −  At  least  18  MΩ   §  Install  fresh  polymer,  buffer  and  water   §  Clean  the  buffer  jar  and  buffer/water/waste  reservoirs   §  Set  up  samples  with  fresh  aliquot  of  HI-­‐DITM  Formamide  and   new  septa   §  Inject  allelic  ladder  and  size  standards  for  10  consecu6ve  runs   to  see  if  data  problems  persist  
    • 9/18/13 | © Life Technologies™ 43 If  Problems  Persist   §  Install  array  port  plug  and  perform  water  wash  4X  with  40ºC  boSled  water     −  Run  water  wash  wizard  through  the  step  that  flushes  the  PDP  with   water   −  At  this  point,  cancel  the  wizard  and  start  it  again   −  Do  this  un6l  the  pump  system  has  been  flushed  4X   §  Install  a  new  array   §  Set  up  instrument  with  high  quality  water  and  different  lot  of  polymer/ buffer   §  Inject  new  lot  of  allelic  ladder  and  size  standards  for  10  consecu6ve  runs  to   see  if  data  improves     −  Set  up  with  different  lot  of  HI-­‐DITM    
    • 9/18/13 | © Life Technologies™ 44 Common  Capillary  Electrophoresis   Instrument  Issues  and  Observa*ons     § Front  –End  Troubleshoo6ng   § Migra6on  Problems   § Avoiding  Bubbles   § Shadow  Peaks  
    • 9/18/13 | © Life Technologies™ 45 Peak  Migra*on  Problems  
    • 9/18/13 | © Life Technologies™ 46 Peak  Migra*on  Problems:  Common  Causes   §  Polymer  on  instrument  for  >7  days   §  Poor/expired  reagents;  poor  quality  water   §  Buffer   −  Not  changed  daily     −  1X  buffer  not  prepared  correctly   −  Incorrect  buffer  level  on  anode  and/or  cathode  side   §  Ambient  room  temperature  fluctua6ons   §  Bubble  in  path  of  EP  current   −  Can  be  in  the  capillary  or  in  the  polymer  tubing/block   §  Contaminant  in  front-­‐end   §  Non-­‐AB®  sample  plates  (injec6on  abnormali6es)   §  Incomplete  filling  of  capillary  with  polymer   −  Check  for  leak  at  pump  fitngs,  detec6on  cell  or  damaged  lower  block   §  Oven  gasket  (addressed  by  AB®  retrofit)  
    • 9/18/13 | © Life Technologies™ 47 Steps  to  Correct  Migra*on  Problems   §  Check  for  polymer  leak  at  PDP  fitngs  and  at  lower  block  pin  valve   §  Be  sure  to  use  high  quality  water  source  for:   −  Cleaning  the  system   −  Making  1X  buffer   −  Filling  the  water/waste  reservoirs   §  Perform  water  wash  and  install  fresh  polymer,  buffer,  water  and  septa   §  Set  up  samples  with  fresh  aliquot  of  HI-­‐DITM  formamide   §  Maintain  consistent  lab  temperature  (20-­‐30ºC)   §  Use  AB®  sample  plates  &  always  spin  down  plates   §  If  problems  persist,  replace  the  array  
    • 9/18/13 | © Life Technologies™ 48 <19°C 24.5oC Influence  of  Environmental  Temperature   Comparison  of  EPT  view  
    • 9/18/13 | © Life Technologies™ 49 Influence  of  Environmental  Temperature   Low  Molecular  Weight  Peak  Morphology   3130xl System: rt = 18 °C 3130xl System: rt = 21°C 3100 System: rt = 23°C
    • 9/18/13 | © Life Technologies™ 50 Common  Capillary  Electrophoresis   Instrument  Issues  and  Observa*ons     § Front  –End  Troubleshoo6ng   § Migra6on  Problems   § Avoiding  Bubbles   § Shadow  Peaks  
    • 9/18/13 | © Life Technologies™ 51 Problems  Ocen  Caused  by  Bubbles   §  Failed  injec6ons   §  Inconsistent  peak  migra6on   §  Spikes  in  data   §  Damage  to  interconnect  tubing  and/or  PDP   §  Lower  block  damage   §  “Leak  detected”  errors   §  Erra6c  EP  current  
    • 9/18/13 | © Life Technologies™ 52 Lower  Block  Damaged  by  Bubbles:  Arcing  
    • 9/18/13 | © Life Technologies™ 53 More  Damaged  Lower  Blocks  
    • 9/18/13 | © Life Technologies™ 54 Erra*c  EP  Current  Due  to  Bubbles  
    • 9/18/13 | © Life Technologies™ 55 Avoiding  Bubbles   §  Warm  &  degas  polymer  prior  to  installing   −  Allow  polymer  to  sit  at  room  temperature  for  at  least  an  hour  with  cap   loosened   §  Changes  in  ambient  temperature  can  result  in  bubble  forma6on   −  Be  sure  the  lab  thermostat  is  set  to  a  consistent  temperature  24  hours  a   day  (20-­‐30ºC)   −  Do  not  place  the  instrument  next  to  hea6ng  or  air  condi6oning  vents   §  Periodically  check  that  all  PDP  fitngs  are  finger-­‐6ght   §  Check  for  bubbles  every  day  in  the  pump  chamber,  tubing,  array  port   §  Use  Bubble  Remove  Wizard  to  get  rid  of  bubbles   §  Stubborn  bubbles  may  require  a  Water  Wash  Wizard  
    • 9/18/13 | © Life Technologies™ 56 Common  Capillary  Electrophoresis   Instrument  Issues  and  Observa*ons     § Front  –End  Troubleshoo6ng   § Migra6on  Problems   § Avoiding  Bubbles   § Shadow  Peaks  
    • 9/18/13 | © Life Technologies™ 57  What  are  Shadow  Peaks?   §  The  ar6fact  peaks  appear  as  “shadow  peaks”  to  true  DNA  peaks  observed  in   the  electropherograms  of  amplified  samples  co-­‐injected  with  GeneScan™  500   ROX™  or  GeneScan™  500  LIZ®  size  standard.   §  In  most  cases,  these  ar6facts  are  most  prevalent  in  the  dye  channel   corresponding  to  the  size  standard  and  do  not  affect  accurate  sizing  of  the  size   standard  peaks.   §  Results  of  inves6ga6ons  performed  at  Applied  Biosystems  to  determine  the   cause  of  the  shadow  peaks  suggest  that  the  peaks  are  caused  by  pre-­‐  and/or   post-­‐injec6on  hybridiza6on.    
    • 9/18/13 | © Life Technologies™ 58 Hypothesis   §  The  extra  peak  is  most  likely  the  non-­‐denatured  form  (dsDNA)  of  the   fragment  (ssDNA)   −  dsDNA  could  migrate  faster  than  ssDNA  for  two  reasons   >  Primary  effect:  dsDNA  has  twice  as  many  nega6ve  charge  than   ssDNA   >  Configura6on  of  dsDNA  makes  faster  migra6on  more  favorable     −  The  sum  of  the  peak  height  (dsDNA  +  ssDNA)  within  a  locus  is  conserved   between  a  non-­‐denatured  and  denatured  sample     General schematic of CE Some fragments are present as dsDNA and travel faster than ssDNA during CE injection. It denatures once it hits the oven.
    • 9/18/13 | © Life Technologies™ 59 Verifica*on  Study   Replacement  of  Water  Wash  with  Buffer    Experiments  have  been  performed  which  demonstrate  the   elimina6on  or  significant  reduc6on  of  the  shadow  peaks  on  Applied   Biosystems®  3130  Gene6c  Analyzers  when  the  water  in  the  water   reservoir  rinse  tray  (tray  4  in  figure  below)  is  replaced  with  1X   Gene6c  Analyzer  Buffer.   Note: The configuration for the 3100 is slightly different. See manual for configuration.
    • 9/18/13 | © Life Technologies™ 60 §  GeneScan™  500  LIZ®  and  GeneScan™  500  ROX™  size  standards   as  well  as  in  samples  amplified  with  the  AmpFlSTR®  Iden6filer®   kit  and  co-­‐injected  with  GeneScan  ™  500  LIZ®  size  standard.     §  Studies  were  performed  to  examine  the  impact  of  the  buffer   replacement:   −  genotype  concordance,  precision,  peak  resolu6on,  intracolor   balance,  and  overall  peak  height   −  AmpFlSTR®  SGM  Plus  and  AmpFlSTR®  Iden6filer®  kits   analyzed  on  3100  and  3130xl  capillary  electrophoresis   instruments.     Verifica*on  Study   Replacement  of  Water  Wash  with  Buffer  
    • 9/18/13 | © Life Technologies™ 61 Results  –  Shadow  Peak  Ra*o   GS500 LIZ Identifiler High Input DNA Note: GS500 LIZ - 20 hours after mixing with HI-DITM; 9947A - 6 ng reaction, 15 hours after mixing with HI-DITM
    • 9/18/13 | © Life Technologies™ 62 Verifica*on  Study   Replacement  of  Water  Wash  with  Buffer   §  Shadow  Peak  Height  Ra6o   −  Undetermined  because  shadow  peaks  were  not  observed   −  Buffer  wash  reduced  peak  height  ra6o  from  2-­‐20%  to  0-­‐2%  (ABJ  MCB)   §  Peak  Height   −  Calculated  rela6ve  change  in  peak  height  from  water  to  buffer  wash   −  Decreased  by  an  average  of  18%   §  Genotype  Concordance   −  100%  concordance  for  samples  with  complete  profile   −  Number  of  incomplete  profiles  increased  with  0.125ng  samples  most  likely  due  to   decrease  in  peak  height   §  Sizing  Precision   −  No  differences  due  to  water  or  buffer  capillary  wash   −  Met  specifica6on;  standard  devia6on  below  0.15bp  for  all  alleles   §  Intracolor  Balance   −  Similar  results  between  water  and  buffer  wash  
    • 9/18/13 | © Life Technologies™ 63 Shadow  Peaks  Are  Not  Restricted  to  the  ILS   §  Not  all  loci  showed  this  problem   §  Data  generated  below  followed  normal  CE  procedures  except  they  have  omiSed  the   denatura6on  step   −  The  customer  solved  the  problem  by  rerunning  and/or  denaturing  the  samples     2 peaks 4 peaks 2 peaks 4 peaks
    • 9/18/13 | © Life Technologies™ 64 Shadow  Peaks  -­‐  Conclusion   §  The  extra  peaks  (shadow  peaks)  observed  are  non-­‐denatured  fragments   §  These  peaks  were  present  due  to  incomplete  denatura6on   §  Not  all  of  the  dye  and  loci  were  affected  –  indica6ng  that  some  fragments  are   harder  to  denature.   §  Shadow  Peaks  generally  appear  first  in  the  size  standard.     §  During  tes6ng,  it  was  generally  found  that  arrays  with  high  number  of  runs   (over  200  runs)  are  more  prone  to  shadow  peak  forma6on.  
    • 9/18/13 | © Life Technologies™ 65 Shadow  Peaks  -­‐  Recommenda*ons   §  The  following  recommenda6ons  can  help  minimize  observa6on  of  the  shadow  peaks:     −  Target  an  appropriate  amount  of  input  DNA.     >  Excess  DNA  loading  on  the  capillary  6p  is  more  likely  to  cause  shadow  peak   forma6on.   >  Exceeding  recommended  DNA:formamide  ra6o  results  in  reduced  denaturing     −  Perform  capillary  electrophoresis  tes6ng  on  freshly  prepared  plates  (<8-­‐24  hours).   −  Ensure  proper  aliquo6ng  and  storage  of  HI  DI™  formamide  to  avoid  uptake  of  water   in  the  formamide.     §  If  shadow  peaks  are  observed  in  a  par6cular  injec6on,  the  following  steps  may  reduce   or  eliminate  the  peaks:     −  Re-­‐inject  the  prepared  sample.     −  If  no  improvement  in  results  is  seen,  re-­‐denature  the  sample  by  hea6ng  to  95°C  for   3  minutes  and  chilling  for  at  least  3  minutes  on  ice  and  re-­‐inject.     −  If  again  no  improvement  is  seen,  re-­‐prepare  the  amplified  product  with  fresh  HiDi™   formamide  and  size  standard,  denature  and  re-­‐inject.  
    • 9/18/13 | © Life Technologies™ 66 Carryover  Introduc*on   §  Customers  have  reported  seeing  carryover  in  3130  Series  Gene6c  Analyzers  in   recent  years   §  Communica6ons  issued  in  July  2010  and  June  2012  reiterated  that  carryover,   among  other  aspects  of  capillary  electrophoresis  (CE)  is  one  of  the  factors  to   consider  when  performing  data  analysis.    (References:  Maximizing  the  Performance  of  Capillary   Electrophoresis  Systems,  Life  Technologies  Forensic  News,  July  2010  and  Considera:ons  for  Evalua:ng  Carryover  on  Applied   Biosystems  Capillary  Electrophoresis  Pla?orms  in  a  HID  Laboratory,  Life  Technologies  Technical  Note,  June,  2012)   §  The  primary  mechanisms  for  minimizing  carryover  are  the  polymer  fill  and  rinse   steps  of  the  run  module,  and  the  duck  bill  design  of  the  septa  to  physically  wipe   the  ends  of  the  capillary.      While  this  has  always  been  the  design  since  the   original  310  Gene6c  Analyzer,  absolute  removal  of  all  PCR  product  cannot  be   guaranteed,  only  minimized.  
    • 9/18/13 | © Life Technologies™ 67 Carryover  Introduc*on   §  “In  contrast  to  crosstalk,  which  affects  only  adjacent  capillaries   within  a  single  injec6on,  carryover  is  defined  as  the  physical   transfer  of  DNA  from  one  injec6on  to  the  next.  Therefore,   carryover  can  affect  both  single  and  mul6-­‐capillary  pla_orms  and,   unlike  a  crosstalk  evalua6on,  the  evalua6on  of  carryover  is   performed  by  evalua6ng  the  subsequent  injec6on  of  a  single   capillary  for  any  sign  of  signal  from  a  previous  injec6on.”  Forensic   News,  July  2010  
    • 9/18/13 | © Life Technologies™ 68 Carryover  Introduc*on   §  Con6nuous  studies  being  performed  from  December,  2012   through  March,  2013  in  Foster  City   §  Studies  at  Life  Technologies  have  been  performed  to  inves6gate   the  following:   −  What  is  the  current  incidence  of  carryover?   >  Large  study  with  many  samples  to  es6mate  expected  frequency  and  level   −  Will  a  beSer  wash  module  help  reduce  carryover?   >  Engineering  approach  to  designing  an  op6mal  wash  module   −  Do  older  reservoir  septa  exhibit  carryover?   >  Compared  2008  to  2012   −  Do  older  arrays  exhibit  carryover?   >  Tested  array  from  2003     IMPORTANT:  Many  studies  used  par*ally  off  scale  data   to  induce  carryover  and  carryover  counts  may  be   inflated  due  to  off  scale  data,  however  percent   carryover  calcula*on  were  based  on  scale  source  data  
    • 9/18/13 | © Life Technologies™ 69 Carryover  Summary   §  Confirmed  carryover  is  part  of  CE  analysis.    We  encourage   laboratories  to  consider  carryover  when  setng  Analy6cal   Thresholds.   §  Confirmed  carryover  using  5  year  old  septa  and  10  year  old  array.   Incidence  of  carryover  is  variable  but  has  no  apparent  correla6on   to  specific  capillaries  or  specific  septa.       §  As  with  all  signal,  instrument  sensi6vity  can  affect  magnitude  of   carryover.    There  is  no  evidence  that  recent  manufacturing   processes  have  ins6gated  the  recent  reports  of  carryover.  
    • 9/18/13 | © Life Technologies™ 70 Carryover  Summary   § Increased  focus  on  calcula6ng  Analy6cal  thresholds   combined  with  improved  baselines  from  next  genera6on   STR  chemistry  may  contribute  to  the  ability  for   customer’s  to  visualize  carryover.   § Carryover  is  generally  <1%  and,  with  on  scale  source   data,  should  be  commonly  less  than  50  RFU  on  a  3130   (50/7000=1%).    Labs  that  are  analyzing  data  below  this   threshold  are  recommended  to  carefully  evaluate  the   impact  on  data  interpreta6on.  
    • 9/18/13 | © Life Technologies™ 71 Future  Plans   §  Inves6ga6ons  ongoing   §  Further  op6mize  a  final  wash  module  for  customers  who  would   like  to  reduce  carryover  events   §  Inves6gate  the  incidence  of  carryover  on  other  instruments.    To   date  no  confirmed  reports  of  carryover  on  the  3500  Gene6c   Analyzer  with  on-­‐scale  data.    The  reservoir  septa  design  is   different  on  the  3500  compared  to  the  3130  Gene6c  Analyzer.  
    • 9/18/13 | © Life Technologies™ 72 Overview   § Trouble  Shoo6ng  Tools   § Common  Capillary  Electrophoresis  Instrument   Issues  and  Observa6ons   § Common  Amplifica6on  Observa6ons    
    • 9/18/13 | © Life Technologies™ 73 Common  Amplifica*on  Observa*ons   §  Ar6facts   −  Incomplete  A  nucleo6de  addi6on  (-­‐A)   −  Dye-­‐labeled  ar6facts   §  Overamplifica6on   §  Par6al  profiles/Imbalanced  profiles   −  Stochas6c  effects   −  Inhibi6on   −  Degrada6on  
    • 9/18/13 | © Life Technologies™ 74 Common  Amplifica*on  Observa*ons   §  Ar6facts   −  Incomplete  A  nucleo6de  addi6on  (-­‐A)   −  Dye-­‐labeled  ar6facts   §  Overamplifica6on   §  Par6al  profiles/Imbalanced  profiles   −  Stochas6c  effects   −  Inhibi6on   −  Degrada6on  
    • 9/18/13 | © Life Technologies™ 75 What  are  Ar*facts?   §  Inherent  anomalies  in  molecular  biology  systems   −  Ar6facts  will  always  exist   −  Can  cause  interpreta6on  issues  for  forensic  samples   §  Two  sources  of  ar6facts   −  PCR-­‐related   >  E.g.  StuSer,  -­‐A  peaks,  Dye-­‐Labelled  molecules   >  Usually  reproducible  (observed  when  sample  is  re-­‐injected)   −  Post  PCR  and  CE  Instrument-­‐related   >  E.g.  Spikes   >  Usually  non-­‐reproducible  (NOT  observed  when  sample  is  re-­‐injected)  
    • 9/18/13 | © Life Technologies™ 76 Addi*on  of  3’  Non-­‐templated  ‘A’  Nucleo*de   §  Inherent  feature  of   AmpliTaq®  enzyme     §  Primers  are  designed  to   maximize  ‘A’  addi6on  rather   than  try  to  prevent  it     §  Post  PCR  incuba6on  at  60oC   provides  more  6me  for  ‘A’   addi6on  
    • 9/18/13 | © Life Technologies™ 77 Incomplete  Addi*on  of  3’  Non-­‐template  ‘A’   Possible  Causes   §  Too  much  input  DNA   −  Not  enough  6me  to  add  ‘A’  to  all  products   −  ‘A’  nucleo6des  may  become  limi6ng   §  Incorrect  thermal  cycling  parameters   −  Failure  to  program  final  60oC  extension  step   §  Use  of  a  non-­‐validated  thermal  cycler/block   §  Thermal  cycler  failure   §  Poor/incorrect  storage  of  enzyme   §  Reduced/altered  reac6on  volumes    
    • 9/18/13 | © Life Technologies™ 78 -­‐A  vs.  Split  Peaks  Due  to  Cold  Temperatures   §  Which  is  –A  and  which  is  caused  by  electrophoresis?   §  Solu6on:  Re-­‐inject   −  If  split  peaks  disappear  upon  re-­‐injec6on  on  another  CE  or  when  room   temperature  is  higher,  problem  is  not  related  to  amplifica6on   §  To  resolve  –A:   −  Discover  source  of  problem  (see  previous  slide)   −  Rec6fy  source  of  problem   −  Re-­‐amplify  under  proper  condi6ons   vs.
    • 9/18/13 | © Life Technologies™ 79 Decreasing  Injec*on  Time   Decreasing  injec6on  6me  does  not  remove  the  –A!   5 sec injection! 1 sec injection!
    • 9/18/13 | © Life Technologies™ 80 Dye-­‐Labeled  Ar*facts   §  Usually  have  abnormal  peak   morphology   §  Most  ar6facts  have  been   characterized  by  Applied   Biosystems  and  published  in   kit  user  manuals   §  With  proper  use  of  kit,   ar6facts  should  remain   below  50  RFUs  
    • 9/18/13 | © Life Technologies™ 81 Dye-­‐Labeled  Ar*facts   Exaggerated  by:   §  Excessive  exposure  to  heat  or  light   Ø  Do  not  denature  longer  than  3-­‐5  minutes   Ø  Increased  60°C  PCR  extension  6me   §  Improper  storage  of  reagents   §  Shipping/handling  issues   §  Use  of  expired  or  degraded  reagents   §  Using  non-­‐validated  PCR  system   §  Reduced/altered  reac6on  volumes   §  Increased  injec6on  6me   §  Using  more  than  the  recommended  PCR  product  for   electrophoresis  
    • 9/18/13 | © Life Technologies™ 82 Common  Amplifica*on  Observa*ons   §  Ar6facts   −  Incomplete  A  nucleo6de  addi6on  (-­‐A)   −  Dye-­‐labeled  ar6facts   §  Overamplifica6on   §  Par6al  profiles/Imbalanced  profiles   −  Stochas6c  effects   −  Inhibi6on   −  Degrada6on  
    • 9/18/13 | © Life Technologies™ 83 Overamplifica*on   §  Presence  of  pull-­‐up  peaks,  increased  stuSer  ra6os,  noise  and  -­‐A  peaks   §  Poor  peak  morphology   §  Compromised  interpreta6on   §  Off-­‐scale  data  flagged  by  GeneMapper®  ID  SoKware  
    • 9/18/13 | © Life Technologies™ 84 Overamplifica*on  –  Possible  Causes   §  Too  much  input  DNA   −  DNA  not  quan6tated  before  addi6on   −  Quan6ta6on  method  inaccurate   >  Results  outside  dynamic  range   −  Ideal  solu6on  is  to  dilute  and  re-­‐quan6tate  prior  to  amplifica6on   >  Refer  to  quan6ta6on  presenta6ons  for  troubleshoo6ng  info   §  Too  many  PCR  cycles   §  Reduced/altered  reac6on  volumes   §  Poor  pipetng   §  Thermal  cycler  not  performing  properly  
    • 9/18/13 | © Life Technologies™ 85 Common  Amplifica*on  Observa*ons   §  Ar6facts   −  Incomplete  A  nucleo6de  addi6on  (-­‐A)   −  Dye-­‐labeled  ar6facts   §  Overamplifica6on   §  Par6al  profiles/Imbalanced  profiles   −  Stochas6c  effects   −  Inhibi6on   −  Degrada6on  
    • 9/18/13 | © Life Technologies™ 86 Par*al  Profiles  &  Stochas*c  Effects   Possible  Causes   §  Insufficient  DNA  input/limited   sample   §  Quan6ta6on   −  DNA  not  quan6tated   −  Inaccurate  quan6ta6on  method   −  Quan6ta6on  method  does  not   provide  an  effec6ve  indica6on  of   amplifica6on  poten6al  (e.g.   Quan6blot)   §  Degraded  or  inhibited  DNA   §  Improperly  stored/expired   reagents   §  Thermal  cycler  not  performing   properly     007 amplified at 1.0ng 007 amplified at 50pg
    • 9/18/13 | © Life Technologies™ 87 Imbalanced  Profiles:  Degrada*on  vs.  Inhibi*on   Both  usually  represented  by  typical  “ski  slope”  effect   Dis*nc*on  requires  close  examina*on  of:   §  Sample  origin  &  extrac6on  method  u6lized   −  Source  (whole  blood,  semen,  6ssue,  etc.)   −  Substrate   −  Environmental  element  exposure   §  Quan6filer  results   −  Inhibitors  produce  IPC  with  higher  than  expected  CT  values   −  Degraded  samples  would  not  produce  unexpected  IPC  results   §  DNA  profile   −  Off-­‐scale  short  amplicon  peaks  usually  indicate  inhibi6on   −  Random  marker  dropout  also  usually  indicates  inhibi6on  
    • 9/18/13 | © Life Technologies™ 88 Sample re-amplified at AB with 50% DNA concentration Still off scale Sample Degraded? PCR Inhibited Imbalanced  Profiles:  Degrada*on  or  Inhibi*on?     Electropherogram from a Crime Lab Off scale peaks Sent Chelex extract of blood from victim to Applied Biosystems
    • 9/18/13 | © Life Technologies™ 89 Imbalanced  Profiles:  PCR  Inhibi*on   Possible  Causes   §  Too  much  input  DNA  (Preferen6al  Amplifica6on)   −  DNA  not  quan6tated  before  addi6on   −  Quan6ta6on  method  inaccurate   >  Refer  to  quan6ta6on  presenta6ons  for  troubleshoo6ng  info   §  Inhibitors  present  in  the  sample   −  Sample  may  require  dilu*on  or  clean-­‐up  to  reduce  level  of  inhibi6on  and   improve  profile  quality   >  Hema*n  and  Heme   >  Soil   >  Indigo  dyes   >  Phenol   >  Etc.  
    • 9/18/13 | © Life Technologies™ 90 Reac*on  Volume   §  Profiler  Plus®  &  COfiler®  kits  are  op:mized    &  validated  with  a  50  µl  reac6on   volume   §  Iden6filer®,  Yfiler®  &  MiniFiler®  kits  are  op:mized  &  validated  with  a  25  µl   reac6on  volume   §  Performance  expecta6ons  and  interpreta6on  guidelines  documented  in  the   AmpFℓSTR®  Kit  User  Manuals  are  based  on  these  validated  reac6on   volumes   −  E.g.  Inter-­‐  and  intra-­‐color  balance;  signal  intensity;  heterozygote  peak  balance;   stuSer  percentage;  capacity  to  cope  with  sample  inhibi6on   §  Quality  Control  evalua6ons  prior  to  kit  release  are  performed  at  the   validated  reac6on  volumes   −  Therefore,  if  your  lab  uses  a  reduced  reac6on  volume,  it  is  possible  that  you   may  see  ar6facts/anomalies  that  we  did  not  
    • 9/18/13 | © Life Technologies™ 91 General  Amplifica*on  Recommenda*ons   §  Do  not  use  any  kit  components  beyond  kit  expira6on  dates   §  Do  not  combine  components  from  different  kit  lots  (kits  are  QC’ed  together  as  a  lot)   §  Do  not  alter  reac6on  volumes  or  thermal  cycling  parameters  from  manufacturer’s   recommenda6ons     §  Store  all  kit  components  appropriately:   >  Primer  set,  reac6on  mix  and  controls  stored  at  2  to  8  °C  (limit  primer  set   exposure  to  light)   >  Taq  Polymerase  stored  at  -­‐15  to  -­‐25  °C     >  Ladders  stored  at  -­‐15  to  -­‐25  °C  for  long  term  storage;  once  in  use,  store  at   2  to  8  °C    
    • 9/18/13 | © Life Technologies™ 92 Troubleshoo*ng  Amplifica*on  Summary   §  To  minimize  the  number  of  amplifica6on  issues:   −  Ensure  the  correct  amount  of  DNA  is  added  to  the  reac6on  through  the  use  of   an  effec6ve  quan6ta6on  technique     −  Follow  manufacturer’s  recommenda6ons  for  amplifica6on  using  the  Applied   Biosystems  Kits   >  Reac6on  volume   >  Input  DNA  concentra6on   >  Use  of  a  validated  thermal  cycler/block   >  Use  of  correct  thermal  cycling  parameters   −  Ensure  thermal  cycler  calibra6ons  and  proper  temperature  verifica6ons  are   performed  rou6nely   §  Unless  otherwise  directed,  use  colorless  tubes/plas6cs  throughout  the  analy6cal   process  as  recommended  by  Applied  Biosystems  
    • Thanks!  
    • 9/18/13 | © Life Technologies™ 94 Legal  Statements    For  Research,  Forensic  or  Paternity  Use  Only.  Not  for  use  in  diagnos6c  procedures.        AB  (Design),  Applied  Biosystems,  GeneMapper,  and  HID  (Design)  are  registered   trademarks  of  Applied  Biosystems  or  its  affiliates  in  the  US  and/or  certain  other   countries.    All  other  trademarks  are  the  sole  property  of  their  respec6ve  owners.      Please  refer  to  the  product  inserts  for  informa6on  on  relevant  patent  coverage.  For   further  informa6on  contact  the  Director  of    Licensing,  Applied  Biosystems,  850  Lincoln   Centre  Drive,  Foster  City,  California  94404,  USA.