Megachile rotundata

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Data

Original Traces

  • 8 pairs of data files (paired ends)
 cat trace.count | grep _1_ | sed 's/_sequence.txt//' | perl -ane 'print "  ",$F[1],"\t",$F[0]/4,"\t",$F[0]/2,"\n";'

 lib        insert   mates           reads        readLen   ~coverage(500M genome)  
 s_2_3kbp   3000     21,563,283      43,126,566   124       11                              
 s_2_8kbp   8000     198377          396,754      124       0.1                             
 s_3        475      35548153        71,096,306   124       18
 s_4        475      35471044        70,942,088   124       18
 s_5        475      35616846        71,233,692   124       18
 s_6        475      35303840        70,607,680   124       18
 s_7        475      34893313        69,786,626   124       18
 total      .        198,594,856     397,189,712  128       98*

Corrected Traces

  • Mated ones
 lib        insert   mates           reads          repeatReads
 s_2_3kb    3000     4,823,235       9,646,470      4,349,208  (45%)
 s_2_8kb    8000     111,267         222,534        167,246    (75%)
 s_3        475      33,024,597      66,049,194     35,777,342 (54%)
 s_4        475      33,237,593      66,475,186     
 s_5        475      33,150,790      66,301,580  
 s_6        475      33,223,371      66,446,742  
 s_7        475      32,647,890      65,295,780
 total      .        170,218,743     340,437,486
  • repeatReads:
    • at least one of the mate contains a perfect match of one of the 15 frequent 22mers listed below
    • 32.5%GC in repeatREads vs ~ 35.5%GC in uniqueReads

Adaptors

 >circularizarion
 CGTAATAACTTCGTATAGCATACATTATACGAAGTTATACGA
 >circularizarion.revcomp
 TCGTATAACTTCGTATAATGTATGCTATACGAAGTTATTACG

Frequent kmers

  • 22mers which seem to appear in tandem
    1  AATCATACAATCACAATCATAC|GTATGATTGTGATTGTATGATT   :  AATCATAC AATCAC|GTGATTGTATGATT   #14mer
    2  CAATCACAATCATACAATCACA|TGTGATTGTATGATTGTGATTG
    3  AATAATATGAGTTAGATTGATA|TATCAATCTAACTCATATTATT
    4  ATATAAGCATAATATGGCTAAT|ATTAGCCATATTATGCTTATAT
    5  AGTAATTGTCGTTCTATCGATC|GATCGATAGAACGACAATTACT
    6  AGACAGAGACAGAGACAGAGAC|GTCTCTGTCTCTGTCTCTGTCT
    7  TCACACAATCACAATCACACAA|TTGTGTGATTGTGATTGTGTGA
    8  CACACAATCACACAATCACACA|TGTGTGATTGTGTGATTGTGTG
    9  ATTACTCTTATTATTATCAATC|GATTGATAATAATAAGAGTAAT
   10  CTGTCTCTGTCTGTCTCTGTCT|AGACAGAGACAGACAGAGACAG
   11  ACAATTACTATACTTATTACTC|GAGTAATAAGTATAGTAATTGT
   12  CACAATCACGATCACACAATCA|TGATTGTGTGATCGTGATTGTG
   13  AACCTAACCTAACCTAACCTAA|TTAGGTTAGGTTAGGTTAGGTT
   14  CAGCGGATATGTGCGAATTAGA|TCTAATTCGCACATATCCGCTG
   15  CTGAGCACAATTCAACACCACA|TGTGGTGTTGAATTGTGCTCAG

Location

 /fs/szattic-asmg5/Bees/Megachile_rotundata/error_correction/large_libs/s_?_?_?kb.sequence.cor.all.txt
 /fs/szattic-asmg5/Bees/Megachile_rotundata/error_free/s_?_?_sequence.cor.txt
 /fs/szattic-asmg5/Bees/Megachile_rotundata/frg/  # frg files to assemble

Assemblies

  • CA Version: 6.1 (09/01/2010) /fs/szdevel/dpuiu/SourceForge/wgs-6.1/Linux-amd64/bin/runCA
  • SOAP version 1.04: /nfshomes/dpuiu/szdevel/SOAPdenovo_Release1.04/

CA noOBT partial

  • Data : 3 libs : ~ 16X cvg

Gatekeeper

 LibraryName           numActiveFRG    numDeletedFRG  numMatedFRG  readLength  clearLength  
 GLOBAL                72,995,448      0              70632632     8307194830  8278360381   
 LegacyUnmatedReads    0               0              0            0           0            
 s_2_3kb               9,166,343       0              8736228      942501164   914798596    
 s_2_8kb               210,266         0              199620       21669112    20742291     
 s_3                   63,618,839      0              61696784     7343024554  7342819494

 UID             IID     mateUID         mateIID libUID  libIID  isDel   isNonRandom     Orient  Length  clrBeginLATEST  clrEndLATEST
 110000000001    1       120000000001    2       s_2_3kb 1       0       0               I       75      0               75
 120000000001    2       110000000001    1       s_2_3kb 1       0       0               I       123     0               123
 110000000003    3       120000000003    4       s_2_3kb 1       0       0               I       90      0               90
 120000000003    4       110000000003    3       s_2_3kb 1       0       0               I       123     40              123
 ...
 110009166343    9166343 0               0       s_2_3kb 1       0       0               U       76      11              76

 210009166344    9166344 220009166344    9166345 s_2_8kb 2       0       0               I       123     21              123
 ...
 210009376609    9376609 0               0       s_2_8kb 2       0       0               U       88      0               88

 320009376610    9376610 0               0       s_3     3       0       0               U       72      0               72
 ...
 310072995448    72995448 0              0       s_3     3       0       0               U       68      0               68

 gatekeeper -b 1 -e 2 -dumpfastaseq asm.gkpStore/  | more
 >110000000001,1 mate=120000000001,2 lib=s_2_3kb,1 clr=LATEST,0,75 deleted=0
 TAATAATATGCGTTAGAGTGATAATAATTGGAGTAATGAGTATAGCAATTGTCGTCCTGTCGATCATATCTGCAT
 >120000000001,2 mate=110000000001,1 lib=s_2_3kb,1 clr=LATEST,0,123 deleted=0
 CCATATTATGCTTATATGATCGATAGAACGACAATTACTATACTTATTATTCTTATTATTATCAATCTCACTCATATTATTAACCATATTATGCAGACATGATCGATAGAACGGCAATTACTA

 head -2 s_2_3kb_?.filter.seq
 >HWI-EAS385_0062:2:1:1036:15608#GCCAAT/1
 TAATAATATGCGTTAGAGTGATAATAATTGGAGTAATGAGTATAGCAATTGTCGTCCTGTCGATCATATCTGCAT
 >HWI-EAS385_0062:2:1:1036:15608#GCCAAT/2
 CCATATTATGCTTATATGATCGATAGAACGACAATTACTATACTTATTATTCTTATTATTATCAATCTCACTCATATTATTAACCATATTATGCAGACATGATCGATAGAACGGCAATTACTA

Stats

 .                  elem       min    q1     q2     q3     max        mean       n50        sum             #repeats    comments          
 scf                20,827     122    3228   6374   13700  202495     11508      20462      239696810                   SOAPdenovo: max=1102803 , N50=26876  
 ctg                37,494     65     2185   3998   7706   191323*    6380       10151*     239226293       206         SOAPdenovo: max=121554  , N50=3138
 deg                1,136,469  64     123    143    184    5031       160        164        181954480       807132
 utg                1,437,146  64     123    143    195    67048      308        870        443759899      

 readsTotal         72,995,448
 readsInContigs     27,837,956
 readsInDegenerates  9,627,122
 singletons         34,881,692 (47%)                                                             31,987,184

 readsWithOuttieMate 3,028,956(4.15%) ???

Issues

  • lib s_2_* orientation ???
 cat asm.posmap.frags | grep placed | p '/(\w)/; print "$1 $F[4]\n";' | count2col.pl | pretty -o
 .    badLong  badOuttie   badSame  bothDegen  bothSurrogate  diffScaffold  good      notMated  oneChaff  oneDegen  oneSurrogate  
 1    534      2,998,286   458      1614846    9892           21872         27308     267328    979980    760044    65268         
 2    4        26,864      10       38636      114            294           178       5044      35465     7848      1022          
 3    11072    3,806       1104     2369982    61236          53370         23058022  1208112   3967689   371538    87260

 cat asm.posmap.frags | grep -v placed | p '/(\w)/; print "$1 $F[4]\n";' | count2col.pl | pretty -o
 .    bothChaff    notMated   oneChaff  
 1    1,277,760    162,787    979,980    
 2    53,588       5,602      35,465     
 3    27,684,878   713,943    3,967,689

Location

 ginkgo:/scratch1/dpuiu/Megachile_rotundata/Assembly/wgs-noOBT-partial/

 Moved  from
 mulberry:/scratch2/dpuiu/Megachile_rotundata/Assembly/wgs-noOBT-partial/

CA noOBT

  • Data : 7 libs : ~ 74X cvg

Gatekeeper

 LibraryName           numActiveFRG  numDelFRG  numMatedFRG  readLength   clearLength    #repeats                                                                                                                                                              
 GLOBAL                326,236,387   0          315518526    37451489553  37418130441  
 LegacyUnmatedReads    0             0          0            0            0            
 s_2_3kb               9107424       0          9107424      942165284    910444046      #
 s_2_8kb               209336        0          209336       21814418     20787384       #
 s_3                   63618839      0          61696784     7343024554   7342819494     #
 s_4                   63544688      0          61255960     7291557748   7291478152     #
 s_5                   63370860      0          61084368     7271218123   7271051639     #
 s_6                   63780887      0          61685156     7359094156   7359012512     #
 s_7                   62604353      0          60479498     7222615270   7222537214     #

Meryl

 meryl -Dh -s 0-mercounts/asm-C-ms22-cm0 
 Found 30570218845 mers.
 Found 271464470 distinct mers.
 Found 11164787 unique mers.
 Largest mercount is 87984949; 1896 mers are too big for histogram.
 1       11164787        0.0411  0.0004
 2       9376915         0.0757  0.0010
 3       3714582         0.0894  0.0013
 ...
 54      5344148         0.6573  0.1788
 ... 

 fasta2tab.pl 0-mercounts/asm.nmers.ovl.fasta | sort -n -r | head -5
 87,908,217        AATCATACAATCACAATCATAC
 84,450,288        CAATCATACAATCACAATCATA
 ...
 74,975,282        AATAATATGAGTTAGATTGATA

 egrep -c 'AATCATACAATCACAATCATAC|GTATGATTGTGATTGTATGATT' *fastq *txt > egrep.count
 mulberry:/scratch2/dpuiu/Megachile_rotundata/Data/error_free/egrep.count

 meryl -Dh -s 0-mercounts/asm-C-ms15-cm0 | head
 Found 32850820919 mers.
 Found 142500876 distinct mers.
 Found 2381895 unique mers.
 Largest mercount is 125816941; 2023 mers are too big for histogram.
 1       2381895 0.0167  0.0001
 2       2325770 0.0330  0.0002
 3       708786  0.0380  0.0003
 ...
 54      1851586 0.4894  0.0671
...

22mer.png 22mer.cumulative.png

Overlap

  • job count :
 cat 1-overlapper/ovlopts.pl | grep ^\"h | wc -l
 924
  • Failures: 709 jobs failed; runCA 6.1 could not restart overlap properly !!!
 cat 1-overlap/overlap*out | grep "^Could not" | sort -u
 Could not malloc memory (1305184948 bytes)

 cat 1-overlapper/*pl | grep ^\"0 | sed 's/"//' | sed 's/\",//' >! 1-overlapper/ovlopts.pl.0
 cat 1-overlapper/*pl | grep ^\"h | sed 's/"//' | sed 's/\",//' >! 1-overlapper/ovlopts.pl.h
 cat 1-overlapper/*pl | grep ^\"-h | sed 's/"//' | sed 's/\",//' >! 1-overlapper/ovlopts.pl.-h

 paste 1-overlapper/ovlopts.pl.* | p 'print "overlap -M 8GB --hashload 0.8 -t 1 -h $F[3]  -r $F[5] -k 22 -k  \
       ./0-mercounts/asm.nmers.ovl.fasta  -o ./1-overlapper/$F[0]/$F[1].ovb.gz ./asm.gkpStore > \
       ./1-overlapper/overlap.0$..out \n";' | tail -709 > overlap.sh
  • Stats
 overlapStore -d asm.ovlStore | awk '{print $1}' | uniq -c | awk '{print $1}' | count.pl | getSummary.pl -i 0 -j 1
 overlapStats -G asm.gkpStore -O asm.ovlStore -o asm

Location

 mulberry:/scratch2/dpuiu/Megachile_rotundata/Assembly/wgs-noOBT

SOAPdenovo (Tanja)

 cat *.ContigIndex | grep -v ^E | grep -v ^i | count.pl -i 1 | getSummary.pl -j 1 -t "contigs"
 cat *.ContigIndex | grep -v ^E | grep -v ^i | count.pl -i 1 | getSummary.pl -j 1 -min 100 -t "contigs(>100bp)"
 grep "^>" *.scaf | getSummary.pl -i 2 -t scaf
  • Stats
 .                    elem       min    q1     q2     q3     max        mean       n50        sum            
 contigs              9742349    31     32     33     37     114832     60         44         585430821      
 contigs(>100bp)      177327     100    131    261    1398   114832     1333       3897       236496823     # N50 for Bee was 7K
 scaf                 7863       102    903    3272   17692  2338728    37825      240706     297423517     # N50 for Bee was 1.17M
  • Location
 /fs/szattic-asmg5/Bees/Megachile_rotundata/Assembly/assembly5kbForAll

SOAPdenovo (Daniela)

  • Stats
 cat asm.K31.contig | grep "^>" | awk '{print $3}' | uniq -c | awk '{print $2,$1}'  > asm.K31.contigLen.count

 .                    elem       min    q1     q2     q3     max        mean       n50        sum            
 contigs(all)         6,917,796  31     32     34     40     121554     70         73         487,401,812      
 contigs(>100bp)      210,666    100    124    222    1174   121554*    1108       3138*      233,563,401
 scaff                25,119     351    1896   4444   10914  1102803    11041      26876      277,338,897

 reads                340,437,486
 readsOnContigs       171,212,613
  • Location
 mulberry:/scratch2/dpuiu/Megachile_rotundata/Assembly/SOAPdenovo-redo
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