Helicobacter pylori

Data

Wustl

• Assemblies
• Assembly tricks
• Article
• Scarf data

NCBI complete genomes

• Genome info

       id             len     gc%
    1  NC_000915.1    1667867 38.87  Helicobacter pylori 26695
    2  NC_000921.1    1643831 39.19  Helicobacter pylori J99
    3  NC_008086.1    1596366 39.08  Helicobacter pylori HPAG1
    4  NC_010698.2    1608548 38.91  Helicobacter pylori Shi470
    5  NC_011333.1    1652982 38.89  Helicobacter pylori G27
    6  NC_011498.1    1673813 38.81  Helicobacter pylori P12
    7  NC_012973.1    1576758 39.16  Helicobacter pylori B38

• nucmer -c 40 => ~200 alignments & 93-95% identity between genomes
• SNPs are mostly substitutions

• Alignment info (NC_000915 0cvg regions) :5-10% of genomes are unique

       .                      elem  min  q1   q2   q3   max    mean  n50   sum
    1  NC_000915-NC_000915    72    45   178  294  1890 10467  1013  1893  72976   #longest alignment has been removed
    2  NC_000915-NC_000921    197   2    81   203  495  17816  644   2146  126988
    3  NC_000915-NC_008086    151   3    103  242  894  26862  951   3103  143652
    4  NC_000915-NC_010698    206   2    115  283  706  12779  726   1941  149744
    5  NC_000915-NC_011333    138   2    111  260  695  7457   688   1941  95063
    6  NC_000915-NC_011498    157   2    83   185  565  5362   505   1357  79337
    7  NC_000915-NC_012973    140   2    108  239  526  37389  1018  5729  142568

• NC_000915 vs NC_000915 : nucmer -c 40

 Align len
.                   elem       min    q1     q2     q3     max        mean       n50        sum
nucmer -c 20        484        20     21     26     82     10467      196        1892       95007
nucmer -c 40        72         45     178    294    1890   10467      1013       1893       72976

 Align %id
.                   elem       min    q1     q2     q3     max        mean       n50        sum
-c 20               484        63.72  91.89  100.00 100.00 100.00     95         100        46014.71
-c 40               72         76.71  85.06  92.86  99.92  100.00     92         93         6639.6

 Media:NC 000915-NC 000915.20.png, Media:NC 000915-NC 000915.40.png

• Repeats (NC_000915.1):

 .                    elem       min    q1     q2     q3     max        mean       n50        sum            
 36                   269        36     45     70     197    4853       305.32     1187       82132          
 100                  112        100    143    267    804    4853       659.07     1891       73816

NCBI SRA

• http://www.ncbi.nlm.nih.gov/sites/entrez?db=sra&term=SRP001104 (24 data sets; 10 not loaded)
• http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?study=SRP001104

Other

• http://msbarker.com/software.htm
• The complete genome sequence of a chronic atrophic gastritis Helicobacter pylori strain: Evolution during disease progression Jun 2006 PNAS
• Response of Gastric Epithelial Progenitors to Helicobacter pylori Isolates Obtained from Swedish Patients with Chronic Atrophic Gastritis

Assemblies

Wustl

velvet contigs 100bp+ stats:

 nl    assembly         ctgs  min  q1   q2    q3     max    mean  n50    sum      reads  0cvg   
 1     HPKX_1039_AG0C1  233   100  318  1700  9639   77743  7085  19912  1650899  6.4m   674288   
 2     HPKX_1039_AG0C2  420   101  341  1684  5301   36588  3914  9412   1644043  5.2M   729736   
 3     HPKX_1039_AG4C1  271   100  217  1421  8273   90368  6115  17093  1657230  6.0m   687296   
 4     HPKX_1039_AG4C2  365   100  301  1595  5658   51523  4522  11890  1650547  6.8M   708607   
 
 5     HPKX_1172_AG0C1  217   107  557  3370  10683  58848  7099  15527  1540507  8.6M   1068848  
 6     HPKX_1172_AG0C2  1170  100  264  717   1768   11444  1319  2661   1543511  7.2M   1110550  
 7     HPKX_1172_AG4C1  377   103  355  2178  6166   35180  4169  9160   1571948  8.5M   1106858  
 8     HPKX_1172_AG4C2  317   100  274  1540  6256   37505  4987  14946  1581161  6.0M   812671   
 
 9     HPKX_1259_NL0C1  1704  100  264  598   1274   7953   936   1606   1595297  4.2m   963211   
 10    HPKX_1259_NL0C2  410   102  240  928   4863   32792  3882  11295  1591864  3.6M   824474   
 11    HPKX_1259_NL4C1  283   100  224  1098  6814   98400  5634  18624  1594699  6.4m   797825   
 12    HPKX_1259_NL4C2  455   102  222  874   4348   32792  3520  11010  1601950  6.3M   833155   
 
 13    HPKX_1379_NL0C1  295   100  230  1243  7551   59556  5539  15858  1634019  6.1m   730236   
 14    HPKX_1379_NL0C2  416   100  216  1000  5177   53581  3931  11219  1635644  6.3M   754915   
 15    HPKX_1379_NL4C1  328   100  227  1084  6601   61090  4996  14203  1638925  5.0m   716540   
 16    HPKX_1379_NL4C2  291   100  231  1501  6751   64227  5539  15080  1612046  4.6M   785276   
 
 17    HPKX_345_AG4C1   251   100  241  1272  8265   97643  6534  19718  1640151  4.5m   727208   
 18    HPKX_345_AG4C2   .     .    .    .     .      .      .     .      .        12.1M  .        
 19    HPKX_345_NL0C1   305   100  243  1146  6718   59632  5360  15874  1634815  5.7m   759194   
 20    HPKX_345_NL0C2   283   100  254  2009  8300   59229  5629  13524  1593064  11.1M  1067288  
 
 21    HPKX_438_AG0C1   267   100  348  1710  8311   87876  6071  16918  1620975  5.8m   755933   
 22    HPKX_438_AG0C2   407   102  396  1777  5455   31183  3963  8830   1613167  6.3M   804474   
 23    HPKX_438_CA4C1   237   100  348  1580  8856   97139  6845  19582  1622487  6.0m   742559   
 24    HPKX_438_CA4C2   485   101  363  1502  4408   35471  3332  7779   1616183  4.1M   801123

CBCB

Summary of what I did to assemble the 24 data sets:

1. I downloaded the read sets and 7 complete related genomes from NCBI  : 36bp unmated reads ; original cvg range: 77-261X cvg : very deep; could be filtered
2. I ran velvet on each read set
3. For the assemblies which were too fragmented and/or had high read coverage I filtered out the low quality reads, ran velvet on the remaining reads and see if the assembly got better. Some assemblies were better some worse.
4. I tried merging the velvet contigs based on direct overlaps (2 contig ends align to each other) or indirect overlaps (2 contigs align to the same complete genome region)
5. Velvet assemblies 6,9,18 were very fragmented so I ran AMOScmp-short reads on all the reads, using as reference the best assembled related HPKX set
6. I tried merging the velvet & AMOScmp contigs based on direct overlaps only

---

Read filtering:

• q00: no reads were filter out
• q20: reads that contained 1+ N's or had avg. quality < 20 were filtered out

Velvet: hash_size=23

velvet contigs 100bp+ stats:

 nl    assembly         ctgs  min  q1   q2    q3     max    mean  n50    sum      reads 0cvg     qual comment
 1b    HPKX_1039_AG0C1  208   101  390  2580  11299  90464  7919  19900  1647226  6.4M  692517   q00  better   
 2     HPKX_1039_AG0C2  417   101  730  2132  5402   34482  3937  8041   1642106  2.9M  722358   q28  similar  
 3     HPKX_1039_AG4C1  202   100  395  2535  12111  82857  8201  20617  1656737  6.0m  683554   q00  similar  
 4     HPKX_1039_AG4C2  250   100  287  2134  9314   90459  6604  18459  1651103  6.7M  703430   q00  better   
 
 5b    HPKX_1172_AG0C1  213   100  229  1521  9559   93714  7423  22783  1581145  7.1M  795376   q20  better   
 6*    HPKX_1172_AG0C2  1122  100  466  900   1639   7935   1243  1857   1395595  3.1M  1915029  q30  worse    
 7     HPKX_1172_AG4C1  313   100  373  2061  7176   67930  5131  13160  1606127  6.0M  811067   q30  better   
 8     HPKX_1172_AG4C2  218   102  280  1561  10678  79935  7225  22481  1575172  5.1M  802832   q20  better   
 
 9*    HPKX_1259_NL0C1  2348  100  242  465   897    8958   678   1061   1593194  4.2m  1060158  q00  worse    
 10    HPKX_1259_NL0C2  271   102  296  1687  7254   78486  5869  17821  1590749  3.1M  811063   q20  similar  
 11b   HPKX_1259_NL4C1  234   102  223  1293  8826   98849  6803  19887  1592056  6.4m  807774   q00  similar  
 12    HPKX_1259_NL4C2  311   101  214  901   6216   78488  5137  17546  1597850  5.5M  816013   q20  similar  
 
 13    HPKX_1379_NL0C1  243   101  212  1087  8703   79083  6723  20883  1633745  6.1m  731779   q00  similar  
 14b   HPKX_1379_NL0C2  237   101  213  1039  8905   79512  6874  22931  1629236  3.9M  744144   q20  similar  
 15    HPKX_1379_NL4C1  257   100  208  764   8197   59755  6382  22923  1640181  5.0m  706386   q00  similar  
 16    HPKX_1379_NL4C2  230   101  225  1440  9348   79083  7055  21887  1622754  4.8M  742039   q20  better   
 
 17b   HPKX_345_AG4C1   254   100  197  1075  8307   88027  6455  20055  1639650  5.5m  739554   q00  worse    
 18*   HPKX_345_AG4C2   1130  101  389  846   1732   14790  1349  2295   1525450  6.5M  1283074  q20  missing  
 19    HPKX_345_NL0C1   253   100  239  1052  8308   75943  6475  19602  1638277  5.7m  744044   q00  better   
 20    HPKX_345_NL0C2   260   101  216  1515  8697   59766  6286  18718  1634563  9.1M  750135   q20  better   
 
 21b   HPKX_438_AG0C1   229   101  358  1701  8830   98294  7075  20804  1620311  5.8m  750029   q00  better   
 22    HPKX_438_AG0C2   272   102  433  2271  7395   53896  5945  14906  1617041  4.7M  768889   q20  better   
 23    HPKX_438_CA4C1   224   102  370  1860  10798  53649  7230  20621  1619628  6.0m  755398   q00  similar  
 24    HPKX_438_CA4C2   356   102  432  2029  6132   36335  4546  11461  1618724  3.0M  775835   q20  better   

 Files:  
    /fs/szasmg3/dpuiu/Helicobacter_pylori/HPKX_*/velvet/

velvet-merged contigs 100bp+ stats: (merged based on alignments to the 7 complete genomes) ; minOVL=5bp

Assembly pipelines:

1. minimus2
   1. ~/bin/AMOS/minimus2:
   2. Align velvet contigs to one another => direct overlaps
   3. Run tigger, make-consensus ...
2. minimus3
   1. ~/bin/AMOS/minimus3:
   2. Align velvet contigs to one another => direct overlaps
   3. Align velvet contigs to all complete reference genomes; find contigs that overlap same reference regions => indirect overlaps
   4. Merge direct & indirect overlaps
   5. Run tigger, make-consensus

 nl    assembly         ctgs min  q1   q2    q3     max     mean   n50    sum       
 1     HPKX_1039_AG0C1  155  101  438  2761  14251  139553  10627  28134  1647320  
 2     HPKX_1039_AG0C2  230  101  575  2665  10199  81516   7129   17062  1639841  
 3     HPKX_1039_AG4C1  169  101  383  2723  13391  126810  9811   25845  1658185  
 4     HPKX_1039_AG4C2  192  100  318  2402  12716  95840   8601   21767  1651530  

 5     HPKX_1172_AG0C1  161  100  229  1602  13093  98369   9826   33745  1582047  
 6*    HPKX_1172_AG0C2  275  100  774  2840  7251   45605   5732   12775  1576564  # merged AMOScmp (ref=HPKX_1172_AG0C1) and velvet contigs; did not use the complete genomes for alignments ; minOVL=40bp
 7     HPKX_1172_AG4C1  204  103  415  2527  12920  76550   7865   19077  1604640  
 8     HPKX_1172_AG4C2  183  102  256  1344  13094  79936   8612   25128  1576072  

 9*    HPKX_1259_NL0C1  249  100  219  837   8006   98866   6485   21851  1614768  # merged AMOScmp (ref=HPKX_1259_NL4C1) and velvet contigs; did not use the complete genomes for alignments ; minOVL=40bp
 10    HPKX_1259_NL0C2  198  102  296  1152  10414  91140   8036   24879  1591324  
 11    HPKX_1259_NL4C1  181  102  259  1136  10406  103908  8801   32108  1593057  
 12    HPKX_1259_NL4C2  255  101  216  729   8686   78488   6272   20602  1599364  

 13    HPKX_1379_NL0C1  191  101  223  976   10495  96103   8565   28132  1635927  
 14    HPKX_1379_NL0C2  183  101  226  1039  11932  96425   8912   32446  1630904  
 15    HPKX_1379_NL4C1  201  101  251  880   9338   100542  8167   27700  1641719  
 16    HPKX_1379_NL4C2  167  101  262  1511  13732  95996   9720   32534  1623349  

 17    HPKX_345_AG4C1   206  101  196  1044  10973  95672   7967   25867  1641285  
 18*   HPKX_345_AG4C2   287  101  341  2032  8153   55325   5683   15371  1631176  # merged AMOScmp (ref HPKX_345_NL0C2) and velvet contigs; did not use the complete genomes for alignments ; minOVL=40bp
 19    HPKX_345_NL0C1   210  100  229  1131  10090  94992   7806   25980  1639377  
 20    HPKX_345_NL0C2   208  101  239  1581  10973  87982   7862   23967  1635408  

 21    HPKX_438_AG0C1   173  102  358  1842  11985  128137  9366   26698  1620475  
 22    HPKX_438_AG0C2   198  102  393  2767  10090  96481   8165   23774  1616761  
 23    HPKX_438_CA4C1   176  102  358  2036  13405  109077  9205   24628  1620172  
 24    HPKX_438_CA4C2   224  102  433  2320  10298  53648   7220   19679  1617399  

 Files:  
    /fs/szasmg3/dpuiu/Helicobacter_pylori/HPKX_*/velvet/minimus3/
    /fs/szasmg3/dpuiu/Helicobacter_pylori/HPKX_*/minimus2/

AMOScmp contigs 100bp+ stats: Pipeline: AMOScmp-shortReads

1. ~/bin/AMOS/AMOScmp-shortReads
   1. Align reads to the "closest" best assembled HPKX reference using soap
   2. Get overlaps
   3. Run casm-layout, make-consensus

 nl    assembly         ctgs min  q1   q2    q3     max     mean   n50    sum     reads  qual   ref   
 2     HPKX_1039_AG0C2  286  100  341  2402  8354   90182   5718   12867  1635626 4.7M   q00    HPKX_1039_AG4C2
 6**   HPKX_1172_AG0C2  367  100  300  1727  5675   37710   4294   10457  1575949 5.5M   q00    HPKX_1172_AG0C1
 9     HPKX_1259_NL0C1  234  103  270  1292  8819   98848   6797   19198  1590643 4.1m   q00    HPKX_1259_NL4C1
 18    HPKX_345_AG4C2   325  101  243  1541  6060   55323   5020   13569  1631537 10.5M  q00    HPKX_345_NL0C2

 Files:  
    /fs/szasmg3/dpuiu/Helicobacter_pylori/HPKX_*/AMOScmp

 Best assembly files:
    /fs/szasmg3/dpuiu/Helicobacter_pylori/HPKX/*
    ftp://ftp.cbcb.umd.edu/pub/data/H_pylori_reassembly/*

• WUSTL assemblies : velvet_?
• CBCB assemblies  : velvet_0.7.55 on Fasta seqs (Fastq: no diffrence )

6 HPKX_1172_AG0C2

Reads:

• all : 7.1M
• q30+: 3.1M
• aligned by soap Helicobacter pylori HPAG1 : 4.8M
• aligned by soap Helicobacter pylori HPKX_1172_AG0C1 : 5.5M

velvet

 .               elem     min  q1   q2   q3    max    mean  n50   sum        reads  0cvg     qual  
 ctgs            1239     45   346  799  1528  7935   1132  1834  1403538    3.1M   1889408  q30   
 ctgs.100+       1122     100  466  900  1639  7935   1243  1857  1395595    3.1M   1915029  q30  

AMOScmp-shortReads (ref HP_HPAG1)

 .               elem     min  q1   q2   q3    max    mean  n50   sum        reads  0cvg     qual
 ctgs.all        1334     36   78   238  1283  16118  1146  3978  1529868    4.8M   1137259  q00
 ctgs.100+       905      100  223  728  2152  16133  1662  4073  1504146    .      .        q00

Directory:

 /fs/szasmg3/dpuiu/Helicobacter_pylori/HPKX_1172_AG0C2.6/AMOScmp.HP_HPAG1

AMOScmp-shortReads (ref 5 HPKX_1172_AG0C1)

 .               elem     min  q1   q2   q3    max    mean  n50   sum        reads  0cvg     qual
 ctgs.all        392      37   227  1470 5481  37710  4024  10457 1577557    5.5M   .        q00
 ctgs.100+       367      100  300  1727 5675  37710  4294  10457 1575949
 
 ref             213      100  229  1521 9559  93714  7423  22783 1581145

Directory:

 /fs/szasmg3/dpuiu/Helicobacter_pylori/HPKX_1172_AG0C2.6/AMOScmp.HPKX_1172_AG0C1

18 HPKX_345_AG4C2

Reads

• 12.1M Solexa 36bp unpaired
• cvg =~ 120X ?

Velvet

Ctg stats :

 hash  #ctgs    min  q1   q2    q3    max    mean  n50    sum
 23    1098     45   244  724   1799  25718  1367  2745   1501014

24 HPKX_438_CA4C2.solexa.txt.assembled-23-11

Reads

• 4.1M Solexa 36bp unpaired
• cvg =~ 80X
• ~9% of the reads contain at least one N

Quality QC:

 .                  elem                                  min    q1     q2     q3     max        mean       n50        sum
 Ncount             4107397    3725799<=0    381598>0     0      0      0      0      35         1          34         5614614
 avgQuality         4107397    118980<=20    3988417>20   0      19     26     29     34         22         28         91690471

 Ncount==0 and avgQuality>=20 => 3013939 filtered reads (73%)

 pos                  elem       min    q1     q2     q3     max        mean       n50        sum
 0                    4107397    0      32     33     33     33         28         33         116108100
 1                    4107397    0      30     33     34     34         27         33         114845690
 5                    4107397    0      27     32     33     34         26         33         109832819
 10                   4107397    0      23     31     33     34         25         32         102860584
 20                   4107397    0      17     28     31     34         22         30         92139539
 30                   4107397    0      2      21     28     34         17         27         70231217
 32                   4107397    0      2      19     26     34         15         26         63156733
 35                   4107397    0      2      2      25     34         13         26         55261361

12mer counts: too much error???

 meryl -C -B -m 12 -s prefix.seq -o prefix.12mers
 meryl -Dh -s prefix.12mers | sort -nk2 -r | more
 1       1876075 0.3452  0.0196
 2       1009161 0.5308  0.0407
 ...
 9       36227   0.7772  0.1017
 10      25866   0.7819  0.1044
 48      20812   0.8729  0.2727  # read cvg ??? 
 49      20726   0.8768  0.2833
 ...

Velvet (all reads)

Ctg stats for different velveth hash_lengths:

 hash  #ctgs    min  q1   q2    q3    max    mean  n50    sum
 19    908      37   161  770   2289  21014  1732  3905   1572704
 21    457      41   84   580   4156  49037  3548  12777  1621652
 23    398      45   161  1435  5137  37278  4068  12278  1619323  (CBCB best*)
 27    769      53   341  1163  2731  18704  2109  4319   1622389

 ?     485      101  363  1502  4408  35471  3332  7779   1616183  (WUSTL)

CBCB best* read cvg =~ 23; repeats at higher cvg

 .     #ctgs    min  q1   q2    q3    max    mean  n50    sum
 cvg   398      13   21   23    25    139    30    25     .

Velvet (filtered reads)

Hash_len=23

Ctg stats

 filter           #ctgs    min  q1   q2    q3    max    mean  n50    sum          #reads     0cvg(all 6 genomes)
 all              398      45   161  1435  5137  37278  4068  12278  1619323      4107397    765345
 noN              420      45   115  1194  4898  37173  3863  11880  1622651      3725799    759343
 avgqual20+       424      45   150  1302  4797  36335  3828  11749  1623136      3069665    757697 
 noN.avgqual20+   453      45   137  1093  4394  36335  3586  11461  1624653      3013939    756829     !!! least seq missing

AMOScmp

Ref : NC_000915

Ctg stats:

 params                           #ctgs      min    q1     q2     q3     max        mean       n50        sum       #readsInCtgs
 -l 16 -c 32 -ovl 10              9533       36     59     96     168    3160       136        185        1302448   1,123,025 (~25%) 1,023,929:0SNP 154,958:1SNP 8,532:2SNP ...
 -l 8  -c 24 -ovl 10              4429       36     62     152    430    5518       350        806        1554095   2,438,762 (~50%) 1,159,669:0SNP 977,687:1SNP 422,738:2SNP ...
 -l 8  -c 24 -ovl 5               3880       36     61     158    492    5883       400        966        1553027   2,438,762 (~50%)

nucmer 0cvg stats:

params                            #gaps      min    q1     q2     q3     max        mean       n50        sum 
 -l 16 -c 32                      8708       2      10     19     41     9286       44         91         388608 
 -l 8  -c 24                      2650       2      8      17     45     2347       56         207        150099

NC_011498

NC_011498.1 1673813 38.81

Reads

• 1.67M Simulated reads 36bp; unpaired; 100% correct;
• the reads were generated by breaking the genome in 36bp segments (35bp ovl)=>36X cvg

Velvet

• Ctg stats :

 hash                     #ctgs    min  q1   q2    q3    max    mean  n50    sum
 23                       292      45   67   164   3422  73108  5654  33268  1651121

Euler-sr

• Ctg stats :

 vertex_size              #ctgs    min  q1   q2    q3    max    mean  n50    sum        #misassemblies  0cvg
 23                       366      24   36   92    931   83748  4596  41745  1682410    . 
 25                       343      26   39   98    1125  83752  5075  42087  1740988    4               27377
 27                       331      28   43  109    1125  83756  5016  41753  1660506    4               27392

AMOScmp

• Ref : NC_000915
• Ctg stats:

 params                                   #ctgs    min  q1   q2    q3    max    mean  n50    sum        #readsInCtgs    misassemblies(<95%length match)
 nucmer -l 16 -c 32 -ovl 10               8569     36   66   114   203   2897   164   231    1405371    836827  (~50%)  39 
 soap -v 5 -g 3 -s 12 -f 2; -ovl 10       1787     37   99   305   1129  14043  881   2283   1574554    1437078 (~85%)  69
 soap -v 5 -g 0 -s 12 -f 2; -ovl 10       1789     37   98   304   1128  14043  880   2283   1574516    1437077         64
 soap -v 3 -g 0 -s 12 -f 2; -ovl 10       3646     36   89   214   532   7184   424   857    1548982                    55
 soap -v 3 -g 0 -s 12 -f 2; -ovl 20       4957     36   81   174   389   4783   316   580    1567357    1353104         49

• 0cvg stats

 params                                   #gaps    min  q1   q2    q3    max    mean  n50    sum
 nucmer -l 16 -c 32 -ovl 10               8026     2    8    15    34    5384   36    75     291959
 soap -v 5 -g 3 -s 12 -f 2; -ovl 10       1042     2    6    19    46    5355   93    746    97176

minimus* on velvet contigs

 .                                    ctgs       min    q1     q2     q3     max        mean       n50        sum            
 velvet                               292        45     67     164    3422   73108      5654       33268      1651121      

                                      ctgs+sing  min    q1     q2     q3     max        mean       n50        sum      misas.
 minimus2(delta-filter -1; OVL=20)    191        45     108    465    10421  117862     8631       33268      1648580  17(6)  # ctgs-vs-ctgs; OVL=20

 minimus3(delta-filter -q; OVL=20)    251        45     68     214    5486   73108      6573       33268      1650065  11()   # ctgs-vs-ref => ctgs-vs-ctgs
 minimus3(delta-filter -q; OVL=5)     191        45     71     227    9222   73108      8631       41743      1648698  6(1)

 minimus3(                OVL=20)     204        45     115    560    10394  117862     8072       33268      1646865  5(1)
 minimus3(                OVL=5)      172        45     134    611    12957  122309     9572       37367      1646538  8(4) 

 minimus3(all ref; OVL=20)            231        45     99     357    7424   117862     7138       33268      1648917  5(1)
 minimus3(all ref; OVL=5)             150        45     140    1177   15439  118850     10959      41743      1643998  15(7)