Works great: ABI PRISM v1.1b2 3700 POP-5 Beta Basecaller

Phillip San Miguel pmiguel at purdue.edu
Tue Sep 5 06:46:52 EST 2000


    I posted some time ago asking whether the new POP-5 Beta
Basecaller[1], etc. was good or not. George Grills wrote me and said
it solved some of the POP-5 mobility shift problems. So I tried it.
    It isn't just a basecaller. It includes a basecaller, a mobility
file and a new dll. Since I had a number of runs still in my 3700
reduced read data base, I changed the mobility files and reextracted
the runs. This was an agonizingly slow and inefficient process.
Disappointingly many of the runs were not reextracted correctly and
gave no useful data. But here are the ones that did work. I used phred
to give quality scores. These are the same runs, extracted first using
the old mobility file and then again with the new mobility file:

             H I G H   Q U A L I T Y   B A S E S
        old mobility    new mobility    improvement
run     mean    median  mean    median  mean    median
___     ______________  ______________  ______________
473     537.3   584     572.4   622     35.1    38
487     512.5   643     541.2   661     28.7    18
486     513.9   637     537.8   657     23.9    20
475     584.9   632     615.6   667     30.7    35
482     270.6   330     292.3   348     21.7    18
483     480     618     505.2   645     25.2    27
454     427.7   504     454     529     26.3    25
453     564.9   635     594.1   656     29.2    21
452     595.5   644     631.1   685     35.6    41
456     533.6   633     555.3   654     21.7    21
455     531.6   638     456.9   527     -74.7   -111
460     488     543     515.5   605     27.5    62
462     553.9   623     585.9   659     32      36
461     556.7   636     492.1   535     -64.6   -101
467     297.8   377     316.6   389     18.8    12
478     382     477     408.3   473     26.3    -4
468     282.6   338     305.2   350     22.6    12
479     445.8   518     151.6   302     -294.2  -216
469     228.2   306     246.8   378     18.6    72
480     399.5   469     422.1   508     22.6    39
481     396.9   508     417.4   540     20.5    32
485     114     218     128.2   204     14.2    -14
488     573.6   635     596     651     22.4    16
489     128.5   244     142     250     13.5    6
451     559.4   618     588.6   640     29.2    22
466     367.2   406     399.5   425     32.3    19
472     592     630     617.8   653     25.8    23
474     585.5   628     611.4   660     25.9    32
476     586.2   612     612.3   636     26.1    24
477     572.2   600     599.6   626     27.4    26
490     586.3   602     614.8   630     28.5    28
471     568.2   644     595.6   680     27.4    36
457     631     664     663.9   695     32.9    31
464     306.5   452     330.9   484     24.4    32
465     187.8   30      205.8   32      18      2
508     563.1   660     594.3   694     31.2    34

    Conclusion? In most cases there was a substantial improvement in
number of phred q>20 bases. But in a few cases there was an alarming
decrease. Perhaps these were actually artifacts of the re-extraction
process. I haven't looked at the individual chromat to find out what
is going on.
    I have done more than 100 runs since changing and the results have
be very good. Indeed it is not uncommon for me to get median numbers
of HQBs over 700. Our best read ever: 805 high quality bases. (Has
anyone else broken 800? We have only done so once--although HQBs in
the 790s appear from time to time.)
    Do the increases in number of phred HQBs represent a true increase
in calling accuracy. I don't know.

Phillip San Miguel
Purdue Genomics Core Facility

[1] Download at:
http://www.appliedbiosystems.com/techsupp/swpps/3700sw.html




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