Megachile rotundata: Difference between revisions

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* Illumina quality scores
* Illumina quality scores
<pre style="background:yellow">
<pre style="background:yellow">
     lib        insert  mates          reads        readLen  ~coverage(500M genome)  adaptor  repeat
     lib        insert  mates          reads        readLen  ~coverage(500M genome)  adaptor  repeat outies
     s_2_3kbp  3000    21,563,283      43,126,566  124      11                      21%,19%  31%
     s_2_3kbp  3000    21,563,283      43,126,566  124      11                      21%,19%  31%     yes
     s_2_8kbp  8000    198,377        396,754      124      0.1                      28%,29%  58%
     s_2_8kbp  8000    198,377        396,754      124      0.1                      28%,29%  58%     yes
     s_2_5kbp  5000    36,218,589      72,437,178  35        5                        no      ?
     s_2_5kbp  5000    36,218,589      72,437,178  35        5                        no      ?       yes
     s_3        475      35,548,153      71,096,306  124      18                      no      50%
     s_3        475      35,548,153      71,096,306  124      18                      no      50%
     s_4        475      35,471,044      70,942,088  124      18                                 
     s_4        475      35,471,044      70,942,088  124      18                                 
Line 19: Line 19:
     s_2_1.4kb  1500    50,861,645      101,723,290  100      20.3                    no      54%
     s_2_1.4kb  1500    50,861,645      101,723,290  100      20.3                    no      54%


     s_7_8kb    8-10kb  25,328,718      50,657,436  125,100  11.4                    21%,13%  39%
     s_7_8kb    8-10kb  25,328,718      50,657,436  125,100  11.4                    21%,13%  39%   yes
     s_7_5kb    5.3kb    29,111,787      58,223,574  36        4.2                      no      23%  
     s_7_5kb    5.3kb    29,111,787      58,223,574  36        4.2                      no      23%   yes          GATCGGAAGAGC
</pre>
</pre>


Line 34: Line 34:
   Password: GRbeehi3
   Password: GRbeehi3


== Qc/Corrected/Addaptor-free Traces ==
== Corrected/Addaptor-free Traces ==


* Sample 10K mates from each lib; compute quality, base composition stats
* Sample 10K mates from each lib; compute quality, base composition stats
   Location:
   Location:
   /nfshomes/dpuiu/Megachile_rotundata/original_sample
   /nfshomes/dpuiu/Megachile_rotundata/original_sample
== Corrected/Addaptor-free Traces ==
=== 1st run (2010 Summer) ===


* Mated ones  
* Mated ones  
Line 51: Line 55:
   s_7        475      32,647,890                65,295,780    36,102,370
   s_7        475      32,647,890                65,295,780    36,102,370
   subTotal  .        170,218,743              340,437,486
   subTotal  .        170,218,743              340,437,486
 
   s_2_1.1kbp  1100    21,502,608              43,005,216    33,900,260
   s_2_1.1kbp  1100    21,502,608              43,005,216    33,900,260
   s_2_1.4kbp  14000    29,125,202              58,250,404    47,076,236
   s_2_1.4kbp  14000    29,125,202              58,250,404    47,076,236
  s_7_8kb    8-10kb    ?                        ?


  s_7_8kb    8-10kb    ?                        ?
* Subset


== Corrected/Adaptor-free Traces (Daniela's selection) ==
   lib                mates                    reads          repeatReads
   lib                mates                    reads          repeatReads
   s_2_3kb.trim64      2,888,124(13.39% orig)    5,776,248      ~0          # these reads aligned to the all read SOAPdenovo assembly; ~ 20% of the mates have 1 mate aligned to linker & ~ 80% of mates are linker free
   s_2_3kb.trim64      2,888,124(13.39% orig)    5,776,248      ~0          # these reads aligned to the all read SOAPdenovo assembly; ~ 20% of the mates have 1 mate aligned to linker & ~ 80% of mates are linker free
Line 70: Line 75:
   /fs/szattic-asmg5/Bees/Megachile_rotundata/error_free/s_?_?_sequence.cor.txt                            # short insert libs   
   /fs/szattic-asmg5/Bees/Megachile_rotundata/error_free/s_?_?_sequence.cor.txt                            # short insert libs   
   /fs/szattic-asmg5/Bees/Megachile_rotundata/frg/                                                        # frg files to assemble
   /fs/szattic-asmg5/Bees/Megachile_rotundata/frg/                                                        # frg files to assemble
=== 2nd run (2011 March) ===
* Mated ones
  lib        insert  mates          mapped  redundant
  s_2_8kb    8000    0
  s_2_5kb    5000    0
  s_3        450      33,044,498
  s_4        450      33,243,183
  s_5        450      33,164,796
  s_6        450      33,227,529
  s_7                32,652,698
  s_2_1.1kb  1100    27,353,742      25.31%  2%
  s_2_1.4kb  1400    43,300,854      20%      3.7%
  s_2_3kb    3000    11,074,463      48.5%    27.3% 
  s_7_5kb    5000    18,954,679      43%      27%
  s_7_8kb    8000    15,101,958      36.6%    35.9%
* Location
  /fs/szattic-asmg5/Bees/Megachile_rotundata/error_correction_3-10/              # re-corrected traces (March 2011)
  ftp://ftp.cbcb.umd.edu/pub/data/assembly/Megachile_rotundata/reads.2011-03/


== Adaptors ==
== Adaptors ==
Line 119: Line 149:
  cat s_2_8k.tab | perl -ane 'print $F[0],"1\n";' >  s_2_1_8k.redundant
  cat s_2_8k.tab | perl -ane 'print $F[0],"1\n";' >  s_2_1_8k.redundant
  cat s_2_8k.tab | perl -ane 'print $F[0],"2\n";' >  s_2_2_8k.redundant
  cat s_2_8k.tab | perl -ane 'print $F[0],"2\n";' >  s_2_2_8k.redundant
== Pacbio data ==
* [[Trace_formatting#Megachile_rotundata_PacBio_sequence]]
* Ftp location:
https://genomexfer.wustl.edu/gxfer1/3535746101561/
beiyohmaifie
cuyogavomaij
* Location
  /fs/szattic-asmg5/Bees/Megachile_rotundata/PacBio
  /fs/szattic-asmg5/Bees/Megachile_rotundata/PacBio/PRIMARY_DATA/*/*fasta                        # 95 files
  /fs/szattic-asmg5/Bees/Megachile_rotundata/PacBio/HQ_READS/*fasta                              # 85 files
  /fs/szattic-asmg5/Bees/Megachile_rotundata/PacBio/FILTERED_TRIMMED_READS/filtered_subreads.fa  # 1 file
* FASTA read length stats
  .                        elem    min  q1    q2    q3    max    mean  n50    sum       
  PRIMARY_DATA              7138674  51    337    550    1054  15128  860    1268  6142469410 
  HQ_READS                  858432  1    470    799    1291  10612  997    1336  855439838 
  FILTERED_TRIMMED_READS    1175261  1    168    517    863    6617    613    934    720454953 
* FASTA read gc% stats
  .                        elem    min  q1    q2    q3    max    mean  n50    sum       
  PRIMARY_DATA              7138674  0.20  54.11  62.95  72.37  98.97  62.97  65.85  .         
  HQ_READS                  858432  0.00  42.18  49.50  60.48  100.00  52.04  53.35  .         
  FILTERED_TRIMMED_READS    1175261  0.00  36.01  42.86  48.44  100.00  41.35  44.80  .
=== 1000 FILTERED_TRIMMED_READS sampled read stats ===
  all                  1000
  bwa -b5 -q2 -r1 -z10  522
  nucmer -l10 -c20      719  # 161 reads have less than 30bp aligned
  nucmer -l10 -c30      439
  nucmer -l15 -c30      433
=== FILTERED_TRIMMED_READS read stats ===


= Assemblies =
= Assemblies =
Line 729: Line 795:
   543.152 188359  3153    #DOWN  2.4:26:1477    2.2:5:1248      #UP
   543.152 188359  3153    #DOWN  2.4:26:1477    2.2:5:1248      #UP
   ...
   ...
=== SOAPdenovo vs CA ===
Aligns contigs 200+bp with nucmer.amos
  set REFN=`grep -c ">" ref.seq`
  @ REFN/=20
  nucmer.amos -D REF=ref -D QRY=qry -D REFN=$REFN ref-qry
  .                  elem      min    q1    q2    q3    max        mean      n50        sum           
  ref(CA)            16507      200    3678  7660  17785  317387    15436      31240      254794314     
  qry(SOAPdenovo)    100877    200    499    1224  2328  111437    2248      4103      226807646     
  cat ref-qry.delta | grep "^>" | sed 's/>//' | awk '{print $1,$3}' | sort -u  | getSummary.pl -i 1
  .                  elem      min    q1    q2    q3    max        mean      n50        sum           
  ref-hits            16195      200    3816  7848  18116  317387    15711      31309      254444709     
  qry-hits            95343      200    610    1307  2427  111437    2356      4171      224616404     
  .                  elem      min    q1    q2    q3    max        mean      n50        sum           
  ref-miss            312        200    244    483    1575  13984      1121      2262      349605       
  qry-miss            5534      200    235    298    442    5658      396        421        2191242


=== Location ===
=== Location ===
Line 749: Line 837:
   contigs100+          214540    100    135    264    1263  122972      1110      2941      238063650
   contigs100+          214540    100    135    264    1263  122972      1110      2941      238063650


== SOAPdenovo vs CA ==
== SOAPdenovo K=31 (new data) ==
* Location
  /scratch1/dpuiu/Megachile_rotundata/Assembly/SOAPdenovo/K31
  ftp://ftp.cbcb.umd.edu/pub/data/assembly/Megachile_rotundata.SOAPdenovo.2011-03/
 
* Assembly stats:
 
  .                elem      min    q1    q2    q3    max        mean      n50        sum
  scf              16115      100    111    134    264    4173260    17307      1288790    278898652
  scfLen            16115      100    111    134    208    3855147    14496      950327    233605903
  ctg              8829648    32    32    34    39    121554    62        3201      553630969
 
  scf2              16115      100    111    134    260    4033560    16539      1067152    266528571
  scf2Len          16115      100    111    134    259    4004254    16236      1071079    261641263
  ctg2              26490      3      120    243    5253  520023    9877      64739      261641355
 
  cat asm.K31.peGrads | tail -6 | p 'print $F[0], " ", $F[1]-$P[1],"\n"; @P=@F' | pretty
  350    330665408 
  1100    54707484 
  1400    86601708 
  3000    22148926 
  5300    37909358 
  8000    30203916 
 
  cat asm.K31.links | awk '{print $5}' | uniq -c  | awk '{print $2,$1}'
  350    7375561
  1100    579996
  1400    604951
  3000    340192
  5300    669339
  8000    184868
 
  7375561 350
  579996 1100
  604951 1400
  340192 3000
  669339 5300
  184868 8000
 
== SOAPdenovo K=47 (new data) ** ==


  Aligns contigs 200+bp with nucmer.amos
<pre style="background:yellow">
  .                    elem      min    q1    q2    q3    max        mean      n50        sum         
  scaf                3495      259    408    757    4763  6,173,378  82382      2,124,089  287,925,734
  scafSeq              31774      100    110    128    172    6,174,792  9215      2,124,853* 292,784,153
  scafSeq2            31774      100    110    128    172    5,876,085  8689      1,814,396  276,082,351
 
  contigs              10217806  48    48    50    56    175,671    77        4,701      792,925,336   
  contigs100+          224315    100    126    177    933    175,671    1134      4,701      254,309,160
  scafSeqContigsClosed 43232      2      114    147    740    479,105    6228      63,194*    269,243,097
</pre>
 
Location:
  /fs/szattic-asmg5/Bees/Megachile_rotundata/Assembly/SOAPdenovo.10libs.K47
 
=== SOAPdenovo vs CA ===
 
              elem      min    q1    q2    q3    max        mean      n50        sum
  CA          16848      64    3501  7433  17428  317387    15124      31240      254808119     
  SO          43232      2      114    147    740    479105    6228      63194      269243097     
 
  CA.no_hits 315        64    124    150    188    1353      168        167        52986         
  SO.no_hits  29679      2      109    123    151    5766      185        158        5489188
 
= Allpaths-LG (experiment) =
 
* Shred SOAPdenovo K=47 contigs >=180bp ; use them as fragment library
 
  .                elem    min  q1    q2    q3    max      mean  n50    sum       
  contig.180+     110580  180  264  964  2321  175671  2166  4701    239535581 
 
* Libraries used: (in_libs.csv)
 
  library_name,    project_name,  organism_name,  type,      paired,  frag_size,  frag_stddev,  insert_size,  insert_stddev,  read_orientation,  genomic_start,  genomic_end  #mates
  frag,            genome,        genome,        fragment,  1,      180,        20,          ,            ,              inward,            0,              0            22,024,446  # originally 176,833,196 fragments insert_size=475bp
  s_2_1.1kb,      genome,        genome,        jumping,  1,      ,          ,            1100,        110,            inward,            0,              0            32,634,858
  s_2_1.4kb,      genome,        genome,        jumping,  1,      ,          ,            1400,        140,            inward,            0,              0            50,861,645
  s_7_5kb,        genome,        genome,        jumping,  1,      ,          ,            5300,        530,            outward,          0,              0            29,111,787
  s_7_8kb,        genome,        genome,        jumping,  1,      ,          ,            8000,        800,            outward,          1,              37            25,328,718
 
  type            #mates.original  #mates.corrected
  frag            22,024,446      21,925,564
  jumping          137,937,008      5,465,197
 
* Assembly stats:
<pre style="background:yellow">
  .                    elem      min    q1    q2    q3    max        mean      n50        sum         
  scf                  1964      1      1187  1550  6679  6,117,056  128,398    1,806,225  252,173,219
  ctg                  36959      1      1836  3140  6343  278,684    6,061      8,784      224,007,758
</pre>
 
= Genbank submission =
 
* [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=143995 Taxonomy]
* [http://www.ncbi.nlm.nih.gov/genomes/mpfsubmission.cgi Project registration]
* [http://www.ncbi.nlm.nih.gov/genomes/mpfsubmission.cgi?show=952ECB8E-7946-4B90-B982-4C2B350C171F Project ID 66515]
* Information
  Project Type: Single Species Project 
  Contacts: Daniela Puiu dpuiu@umiacs.umd.edu, Steven Salzberg salzberg@umiacs.umd.edu
  Submitting Organization: University of Illinois & University of Maryland
  Sequencing Center: Keck Center for Comparative and Functional Genomics, University of Illinois  BCUI  Biotechnology Center, Univ. Illinois (BCUI)
  Consortium Name: Alfalfa Leafcutter Bee Genome Consortium
  Organism Name: Megachile rotundata
  Strain/isolate/breed: North American commercial strain
  Locus Tag Prefix: MROT #3+ letters
  Source of DNA used for sequencing: whole body, haploid brother males
  Sequencing Method: wgs 
  Sequencing Technology: Illumina
  Estimated Genome Size: 250Mb # the haploid genome size
  Brief description of the importance: Megachile rotundata, alfalfa leafcutting bee, is a solitary bee species. It is the #3 agricultural pollinator in the United States and is commercially managed for alfalfa seed production.
  Comments to the staff:
  DNA
  whole genome sequencing
  single genome;
  no annotation
  assembly name MROT_1.0
  assembly method: SOAPdenovo Assembler v_1.05
  plan to update: ?
  expect to release : ?
  strain information to be submitted soon
  genome coverage: 300x
  sequencing technology:  Illumina GA IIx  ??
  Author list:
          Gene E. Robinson,   
          Hugh M. Robertson, 
          Matthew E. Hudson, 
          Kim Walden,         
          Brielle J. Fischman,
          Theresa Pitts-Singer, 
          Rosalind James
          Steven Salzberg,   
          Daniela Puiu,       
          Tanja Magoc,       
          David Kelley,       
          Aleksey Zimin ,     
 
* Best assembly : SOAPdenovo K=47
  /fs/szattic-asmg5/Bees/Megachile_rotundata/Assembly/SOAPdenovo.10libs.K47/genbank[1-3]
 
* Sequence length statistics [1]:
                      number    min    max        mean      n50        sum         
  scaffolds            31774      100    5876085    8689      1814396    276082351   
  contigs              42781      100    479105    6293      69010      269224383
 
* Removed 6 contaminants & scaff < 200bp [2]


   set REFN=`grep -c ">" ref.seq`
   .                   elem      min    max        mean      n50        sum           
   @ REFN/=20
   scaffolds            6367      200    5876085    42857      1699680    272873468   
   nucmer.amos -D REF=ref -D QRY=qry -D REFN=$REFN ref-qry
   contigs              17374      100    479105    15311      64153      266015500


* NCBI comments: '' There are 668 contigs that are <200bp remaining. Some of these  are internal components of scaffolds, but many of these are at the ends of scaffolds so should be removed. For example, 249 of them are the first or component of a scaffold or the only component of a singleton scaffold (list.ShortTerminalContigs).  And some of them are the only components of a multi-component scaffold, eg these two scaffolds are made entirely of short contigs ... ''


   .                 elem      min    q1    q2    q3    max        mean      n50        sum             
   .                   elem      min    max        mean      n50        sum             
   ref(CA)           16507      200    3678  7660  17785  317387    15436     31240      254794314      
   scaffolds           6266      200    5876085    43514     1814396    272660569     # 101 scaffolds deleted
   qry(SOAPdenovo)    100877    200    499    1224  2328  111437     2248      4103      226807646      
   contigs              16706      200    479105     15917      69010      265916502     # 668 contigs deleted


* GPID Organism name Accession
  -----------------------------------------------
  66515 Megachile rotundata AFJA00000000


  cat ref-qry.delta | grep "^>" | sed 's/>//' | awk '{print $1,$3}' | sort -u | getSummary.pl -i 1
  Please cite the accession number as usual:
  .                  elem      min    q1    q2    q3    max        mean      n50        sum           
  ref-hits            16195      200    3816  7848  18116  317387    15711      31309      254444709     
  qry-hits            95343      200    610    1307  2427  111437    2356      4171      224616404     


  .                  elem      min    q1     q2     q3     max        mean      n50        sum           
     This Whole Genome Shotgun project has been deposited at
  ref-miss            312        200    244    483    1575  13984      1121      2262      349605       
     DDBJ/EMBL/GenBank under the accession AFJA00000000.
  qry-miss            5534      200    235    298    442    5658      396        421        2191242
     The version described in this paper is the first version,
    AFJA01000000.

Latest revision as of 20:22, 21 July 2011

Data

Original Traces

  • Illumina quality scores
    lib        insert   mates           reads        readLen   ~coverage(500M genome)   adaptor  repeat  outies
    s_2_3kbp   3000     21,563,283      43,126,566   124       11                       21%,19%  31%     yes
    s_2_8kbp   8000     198,377         396,754      124       0.1                      28%,29%  58%     yes
    s_2_5kbp   5000     36,218,589      72,437,178   35        5                        no       ?       yes
    s_3        475      35,548,153      71,096,306   124       18                       no       50%
    s_4        475      35,471,044      70,942,088   124       18                                
    s_5        475      35,616,846      71,233,692   124       18 
    s_6        475      35,303,840      70,607,680   124       18
    s_7        475      34,893,313      69,786,626   124       18
    subTotal   .        234,813,445     469,626,890   .        98*
 
    s_2_1.1kb  1100     32,634,858      65,269,716   100       13                       no       53%       
    s_2_1.4kb  1500     50,861,645      101,723,290  100       20.3                     no       54%

    s_7_8kb    8-10kb   25,328,718      50,657,436   125,100   11.4                     21%,13%  39%    yes
    s_7_5kb    5.3kb    29,111,787      58,223,574   36        4.2                      no       23%    yes          GATCGGAAGAGC
  • Location
 /fs/szattic-asmg5/Bees/Megachile_rotundata/
 /fs/szattic-asmg5/Bees/Megachile_rotundata/newLibrary/
 /fs/szattic-asmg5/Bees/Megachile_rotundata/newLibrary2/
  • Ftp
 ftp.biotec.illinois.edu 
 ftp://username@ftp.biotec.illinois.edu 
 login: generobi
 Password: GRbeehi3

Corrected/Addaptor-free Traces

  • Sample 10K mates from each lib; compute quality, base composition stats
 Location:
 /nfshomes/dpuiu/Megachile_rotundata/original_sample

Corrected/Addaptor-free Traces

1st run (2010 Summer)

  • Mated ones
 lib        insert   mates                     reads          repeatReads
 s_2_3kb    3000     4,823,235 (22%orig)       9,646,470      4,349,208  (45%)
 s_2_8kb    8000     111,267   (56%orig)       222,534        167,246    (75%) 
 s_2_5kb    5000     36,218,589                72,437,178     35                      # same as original
 s_3        475      33,024,597(92%orig)       66,049,194     35,777,342 (54%)
 s_4        475      33,237,593                66,475,186     36,247,656
 s_5        475      33,150,790                66,301,580     36,350,706
 s_6        475      33,223,371                66,446,742     36,102,470
 s_7        475      32,647,890                65,295,780     36,102,370
 subTotal   .        170,218,743               340,437,486

 s_2_1.1kbp  1100     21,502,608               43,005,216     33,900,260
 s_2_1.4kbp  14000    29,125,202               58,250,404     47,076,236

 s_7_8kb    8-10kb    ?                        ?
  • Subset
 lib                 mates                     reads          repeatReads
 s_2_3kb.trim64      2,888,124(13.39% orig)    5,776,248      ~0          # these reads aligned to the all read SOAPdenovo assembly; ~ 20% of the mates have 1 mate aligned to linker & ~ 80% of mates are linker free
 s_2_8kb.trim64      4,883(0.01% orig)         9,766          ~0          # these reads aligned to the all read SOAPdenovo assembly
  • 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
  • Location
 /fs/szattic-asmg5/Bees/Megachile_rotundata/error_correction/large_libs/s_?_?_?kb.sequence.cor.all.txt   # large insert libs ;  inverted compared to the original (outies => innies)
 /fs/szattic-asmg5/Bees/Megachile_rotundata/error_free/s_?_?_sequence.cor.txt                            # short insert libs  
 /fs/szattic-asmg5/Bees/Megachile_rotundata/frg/                                                         # frg files to assemble

2nd run (2011 March)

  • Mated ones
 lib        insert   mates           mapped   redundant
 s_2_8kb    8000     0
 s_2_5kb    5000     0

 s_3        450      33,044,498
 s_4        450      33,243,183
 s_5        450      33,164,796
 s_6        450      33,227,529
 s_7                 32,652,698

 s_2_1.1kb   1100    27,353,742      25.31%   2%
 s_2_1.4kb   1400    43,300,854      20%      3.7%

 s_2_3kb     3000    11,074,463      48.5%    27.3%   

 s_7_5kb     5000    18,954,679      43%      27%
 s_7_8kb     8000    15,101,958      36.6%    35.9%
  • Location
 /fs/szattic-asmg5/Bees/Megachile_rotundata/error_correction_3-10/              # re-corrected traces (March 2011)
 ftp://ftp.cbcb.umd.edu/pub/data/assembly/Megachile_rotundata/reads.2011-03/

Adaptors

!!! 5' & 3' linkers different than the Bumblebee ones. 

 >circularizarion
 CGTAATAACTTCGTATAGCATACATTATACGAAGTTATACGA
 >circularizarion.revcomp
 TCGTATAACTTCGTATAATGTATGCTATACGAAGTTATTACG
 >5
 CGATAACTTCGTATAATGTATGCTATACGAAGTTATTA
 >3
 GCATAACTTCGTATAGCATACATTATACGAAGTTATACGA

Frequent kmers

  • 22mers which seem to appear in tandem
                                                            ~ %reads    
                                                       s_2_3kb s_2_8kb s_[4567]  s_2_1k
                                                       -------------------------
    1  AATCATACAATCACAATCATAC|GTATGATTGTGATTGTATGATT   12.04   20.1    9.99      21.18
    2  CAATCACAATCATACAATCACA|TGTGATTGTATGATTGTGATTG   10.5    17.87   8.3       2.33
    3  AATAATATGAGTTAGATTGATA|TATCAATCTAACTCATATTATT   7.94    11.77   21.47     9.12
    4  AGTAATTGTCGTTCTATCGATC|GATCGATAGAACGACAATTACT   5.08    7.47    13.04     6.33
    5  ATATAAGCATAATATGGCTAAT|ATTAGCCATATTATGCTTATAT   5.01    7.55    15.15     4.19
    6  CACACAATCACACAATCACACA|TGTGTGATTGTGTGATTGTGTG   4.72    8.57    2.32
    7  ATTACTCTTATTATTATCAATC|GATTGATAATAATAAGAGTAAT   4.62    6.67    11.8
    8  TCACACAATCACAATCACACAA|TTGTGTGATTGTGATTGTGTGA   3.76    7.01    1.54
    9  ACAATTACTATACTTATTACTC|GAGTAATAAGTATAGTAATTGT   2.94    4.39    8.46
   10  AGACAGAGACAGAGACAGAGAC|GTCTCTGTCTCTGTCTCTGTCT   2.17    5.66    1.03      1.03
   11  CACAATCACGATCACACAATCA|TGATTGTGTGATCGTGATTGTG   1.43    2.25    0.5
   12  CTGTCTCTGTCTGTCTCTGTCT|AGACAGAGACAGACAGAGACAG   1.34    3.77    0.68
   13  CAGCGGATATGTGCGAATTAGA|TCTAATTCGCACATATCCGCTG   0.8     0.54    0.73
   14  CTGAGCACAATTCAACACCACA|TGTGGTGTTGAATTGTGCTCAG   0.58    0.35    0.68
   15  AACCTAACCTAACCTAACCTAA|TTAGGTTAGGTTAGGTTAGGTT   0.06    0.15    0.03
   total                                               31      55      50        52
  • Location
 ginkgo:/scratch1/dpuiu/Megachile_rotundata/Data/error_free/noRepeats/         # repeat free FASTQ reads & FRG files
 /fs/szattic-asmg5/Bees/Megachile_rotundata/error_free_repeats/                # repeat ids & unique FASTQ reads

Linker removal

 /fs/szdevel/dpuiu/removeLinkerFromMates/

Identify duplicates

paste s_2_?_8kb*.txt | perl -ane 'chomp; if($.%4==1) { s/\@(\S+)[12]//; printf("%-45s",$1); } elsif($.%4==2) { print substr($F[0],0,32),"\t",substr($F[1],0,32),"\n" } ' | sort -k2,3 | ./getDuplicates.pl > s_2_8k.tab
cat s_2_8k.tab | perl -ane 'print $F[0],"1\n";' >  s_2_1_8k.redundant
cat s_2_8k.tab | perl -ane 'print $F[0],"2\n";' >  s_2_2_8k.redundant

Pacbio data

  • Ftp location:
https://genomexfer.wustl.edu/gxfer1/3535746101561/
beiyohmaifie 
cuyogavomaij
  • Location
 /fs/szattic-asmg5/Bees/Megachile_rotundata/PacBio
 /fs/szattic-asmg5/Bees/Megachile_rotundata/PacBio/PRIMARY_DATA/*/*fasta                        # 95 files
 /fs/szattic-asmg5/Bees/Megachile_rotundata/PacBio/HQ_READS/*fasta                              # 85 files
 /fs/szattic-asmg5/Bees/Megachile_rotundata/PacBio/FILTERED_TRIMMED_READS/filtered_subreads.fa  # 1 file
  • FASTA read length stats
 .                         elem     min   q1     q2     q3     max     mean   n50    sum         
 PRIMARY_DATA              7138674  51    337    550    1054   15128   860    1268   6142469410   
 HQ_READS                  858432   1     470    799    1291   10612   997    1336   855439838   
 FILTERED_TRIMMED_READS    1175261  1     168    517    863    6617    613    934    720454953
  • FASTA read gc% stats
 .                         elem     min   q1     q2     q3     max     mean   n50    sum         
 PRIMARY_DATA              7138674  0.20  54.11  62.95  72.37  98.97   62.97  65.85  .           
 HQ_READS                  858432   0.00  42.18  49.50  60.48  100.00  52.04  53.35  .           
 FILTERED_TRIMMED_READS    1175261  0.00  36.01  42.86  48.44  100.00  41.35  44.80  .

1000 FILTERED_TRIMMED_READS sampled read stats

 all                   1000
 bwa -b5 -q2 -r1 -z10  522 
 nucmer -l10 -c20      719   # 161 reads have less than 30bp aligned
 nucmer -l10 -c30      439 
 nucmer -l15 -c30      433

FILTERED_TRIMMED_READS read stats

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 s_2_3kb, s_2_8kb, s_3

  • Data : 3 libs : ~ 16X cvg
  • Files
 /fs/szattic-asmg5/Bees/Megachile_rotundata/Assembly/assemblyAdaptorFree/longLibrariesAdaptorFree/s_2_?kb_?.filter.fastq   # inverted 
 /fs/szattic-asmg5/Bees/Megachile_rotundata/error_free_better/s_3_?_sequence.cor.txt

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

BOG/ tigStore

  • Number of tigs in the store
 tigStore -g asm.gkpStore -t asm.tigStore 2 -D unitiglist | tail -1 | awk '{print $1}'               # 36318422
  • Single read tigs
 tigStore -g asm.gkpStore -t asm.tigStore 2 -U -d layout | grep -c '^data.num_frags            1$'   # 34985292
 ts2lay | grep -B 9 -A 3 '^data.num_frags            1$'

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%)                                                             

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

 Placed reads
 .          badLong  badOuttie   badSame  bothDegen  bothSurrogate  diffScaffold  good      notMated  oneChaff  oneDegen  oneSurrogate  
 s_2_3kb    534      2,998,286   458      1614846    9892           21872         27308     267328    979980    760044    65268         
 s_2_8kb    4        26,864      10       38636      114            294           178       5044      35465     7848      1022          
 s_3        11072    3,806       1104     2369982    61236          53370         23058022  1208112   3967689   371538    87260

 Chaff reads
 .          bothChaff    notMated   oneChaff  
 s_2_3kb    1,277,760    162,787    979,980    
 s_2_8kb    53,588       5,602      35,465     
 s_3        27,684,878   713,943    3,967,689

Issues

  • reads are renamed : HWI-EAS385_0062:2:1:1036:15608#GCCAAT/1 => UID:110000000001 => IID:1
  • reads < 64bp are deleted from the beginning : ID mapping ???
  • lib s_2 orientation ??? Too many badOuttie's

Location

 ginkgo:/scratch1/dpuiu/Megachile_rotundata/Assembly/wgs-noOBT-partial.1/

CA noOBT ; partial : s_2_3kb, s_2_8kb, s_3 ; no repeats

  • Reads that contain at least one of the 15 most frequent 22mers are deleted from the input set

Gatekeeper

 LibraryName           numActiveFRG  numDeletedFRG  numMatedFRG  readLength  clearLength  
 GLOBAL                33811292      0              32385550     3781368484  3772837304   
 LegacyUnmatedReads    0             0              0            0           0            
 s_2_3kb               5103461       0              4928188      518415011   510187775    
 s_2_8kb               53215         0              51436        5395700     5289866      
 s_3                   28654616      0              27405926     3257557773  3257359663

Overlapper

  • Dirty 3' ends for the s_2_* reads
               totalOvl  avgOvl
 s_2_3kb  5'  4955294   9   
 s_2_3kb  3'  4955294   7   
 s_2_8kb  5'  51050     10  
 s_2_8kb  3'  51050     7   
 s_3      5'  27721948  9   
 s_3      3'  27721948  9

Bog

 cat 4-unitigger/asm.cga.0 | head
 Global Arrival Rate: 0.125220
 There were 1,983,199 unitigs generated.
 Unitig Length
 65407 -  67872:       4 
 50209 -  58608:       5 
 40073 -  49263:      27 
 30132 -  39913:      72 
 20030 -  29892:     319 
 10001 -  19992:    1979 
  9007 -   9999:     673 
  8000 -   8999:     934 
  7000 -   7999:    1332 
  6000 -   6998:    2048 
  5000 -   5999:    3103 
  4000 -   4999:    4898 
  3000 -   3999:    8120 
  2000 -   2999:   14634 
  1000 -   1999:   26621 
   900 -    999:    4116 
   800 -    899:    4457 
   700 -    799:    5042 
   600 -    699:    6146 
   500 -    599:    8107 
   400 -    499:   11901 
   300 -    399:   19373 
   200 -    299:   64394 
   100 -    199: 1173219 
    90 -     99:  161987 
    80 -     89:  189874 
    70 -     79:  132943 
    64 -     69:   82098

UTG

  • The default unitigger tried as well. Fails with the following message:
 unitigger: AS_FGB_io.C:338: void add_overlap_to_graph(Aedge, Tfragment*, Tedge*, IntFragment_ID*, VarArrayIntEdge_ID*, int, int, int, IntEdge_ID*, IntEdge_ID*, IntEdge_ID*): Assertion `ialn > iahg' failed.
 Failure message:
 failed to unitig

Stats

  • Larger max scf & ctg  !!! (compared with "CA noOBT partial" that assembled the repeats as well)
 .                    elem       min    q1     q2     q3     max        mean       n50        sum            
 scf                  21041      65     3174   6334   13482  337719*    11376      20153*     239366537      
 ctg                  37668      65     2181   3963   7687   191376*    6343       10083*     238928665      
 deg                  380596     64     107    126    170    4688       163        160        62151395       # ~22% of deg align with 1 mismatch to kmers
 utg                  652051     64     115    133    225    67870      491        2469       320381694    

 readsTotal           33,811,292
 readsInContigs       27,753,101 (82.08%)
 readsInDegenerates   4,004,853  (11.84%)
 singletons           1,276,811  (3.78%)    # about 12% of singletons align with 1 mismatch to the frequent kmers

 Placed reads
 .    badLong  badOuttie  badSame  bothDegen  bothSurrogate  diffScaffold  good      notMated  oneChaff  oneDegen  oneSurrogate  
 1    582      2992742    410      773006     13486          20146         26870     159957    107838    753916    78412         
 2    1228     2          9486     84         124            62            1550      12940     7750      860       
 3    11354    3884       1074     2266364    90824          56066         23001316  1165421   346169    416116    156218        

 ~/bin/asm2mdi.pl < asm.asm
 s_2_3kb 16      87  ???
 s_2_8kb 8000    800
 s_3     337     27

Location

 ginkgo:/scratch1/dpuiu/Megachile_rotundata/Assembly/wgs-noOBT-partial.1.noRepeats/

CA noOBT ; partial : s_2_3kb & s_2_8kb (trim 64), s_3 no repeats

s_2_3kb & s_2_8kb reads filtering:

  • trimmed to the first 64bp
  • aligned to one of the SOAPdenovo assemblies (soap2)
  • filtered only the mated reads and the single reads with mates aligned to different scaffolds.

The main goal was to get rid of the short inserts present in the long insert libraries (confuse the Celera scaffolder). At the same time I got rid of the linkers (since the linker did not get assembled by SOAPdenovo & all these reads align to the assembly) ... 


 LibraryName         numActiveFRG  numDeletedFRG  numMatedFRG  readLength  clearLength  
 GLOBAL              34220457      0              32793056     3613771597  3613573487   
 s_3                 28654616      0              27405926     3257557773  3257359663   
 s_2_1_3kb.trim64    5556371       0              5377908      355607744   355607744    
 s_2_1_8kb.trim64    9470          0              9222         606080      606080

Stats

 .                elem        min    q1     q2     q3     max         mean       n50        sum            
 scf              6,407       70     3609   10907  37417  854,387     38538      126946     246918298      
 ctg              46,442      64     882    2719   6385   184,930     5235.23    11002      243134341      
 deg              217,175     64     109    134    187    17,896      174.34     184        37861920       
 utg              564,404     64     77     124    226    82,947      539.31     3168       304389506      
 singletonReads   1,125,209

Location

 ginkgo:/scratch1/dpuiu/Megachile_rotundata/Assembly/wgs-noOBT.partial.1.trim64/

CA noOBT ; partial : s_2_3kb, s_2_8kb, s_3 ; no repeats; reverse

  • Reads that contain at least one of the 15 most frequent 22mers are deleted from the input set
  • s_2_3kb & s_2_8kb libraries were reversed (since most of the reads in "CA noOBT partial" were outies)
  • fewer bad mates
  • Smaller contigs & scaffolds

CA noOBT ; partial : s_2_3kb, s_2_8kb, s_3 ; no repeats; no bad links

  • Reads that contain at least one of the 15 most frequent 22mers are deleted from the input set
  • s_2_3kb & s_2_8kb libraries : all mates listed as bad got "broken"

CA OBT ; partial : s_2_3kb, s_2_8kb, s_3 ; no repeats ; doDeduplication

  • smaller contigs, scaffolds

CA noOBT ; partial s_3 , s_4 ; no repeats

  • Reads that contain at least one of the 15 most frequent 22mers are deleted from the input set

Gatekeeper

 LibraryName           numActiveFRG  numDeletedFRG  numMatedFRG  readLength  clearLength  
 GLOBAL                56839966      0              54032986     6425669702  6425397526   
 LegacyUnmatedReads    0             0              0            0           0            
 s_3                   28654616      0              27405926     3257557773  3257359663   
 s_4                   28185350      0              26627060     3168111929  3168037863

Stats

 .                    elem       min    q1     q2     q3     max        mean       n50        sum            
 scf                  12908      148    4003   8811   22202  511831     19416      42207      250623581      
 ctg                  23116      64     2888   5959   13088  255301     10828      20109      250302752      
 deg                  274961     64     124    139    182    4652       172        164        47499725       
 utg                  574873     64     123    132    202    128547     563        4478       324059992

 .      elem     min      q1      q2    q3    max  mean   n50   sum       
 SLK    243      -50905   -7731   -448  -152  126  -5863  126   -1424832  
 CLK    516105   -245237  -59     36    82    208  -1112  208   -574243691  
 ULK    1123683  -150489  -83     6     71    209  -329   209   -369827832  
 CTP    10012    -19762   -20     -9    10    9876 -2     9876  -25270

Location

 ginkgo:/scratch1/dpuiu/Megachile_rotundata/Assembly/wgs-noOBT-partial.2.noRepeats

CA noOBT ; partial : s_2_3kb & s_2_8kb (trim 64), s_3 ,s_4 no repeats

  .                  elem       min    q1     q2     q3     max        mean       n50        sum            
  scf                5159       64     3501   9986   45329  1493864    49143.80   180907     253532854      
  ctg                23598      64     1641   4533   12544  298755     10661.13   26042      251581405      
  deg                258950     64     121    133    176    13861      162.26     161        42018113       
  utg                656004     64     84     124    162    128674     499.73     5935       327823531
 
  scfSeqContigs      7507       6      3643   10925  40664  567232     33678.11   93144      252821608      # gaps closed by SOAPGapCloser

Location: 

 ginkgo:/scratch1/dpuiu/Megachile_rotundata/Assembly/wgs-noOBT-partial.2.noRepeats.trim64/

CA noOBT **

  • Data : 7 libs : ~ 74X cvg

Gatekeeper

 LibraryName           numActiveFRG  numDelFRG  numMatedFRG  readLength   clearLengt #repeats                                                                                                                                      
 GLOBAL                326,236,387   0          315518526    37451489553  37418130441  
 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
 ... 
 87984949 1                                #  AATCATACAATCACAATCATAC

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)
  • Only ~ 60% of the reads had overlaps

Bog

 cat 4-unitigger/asm.cga.0
 Global Arrival Rate: 0.443659
 There were 158,805,551 unitigs generated.
 Unitig Length
Global Arrival Rate: 0.443659       # ???  <=> 200X cvg
100071 - 168549:        21 
 90845 -  99102:        15 
 80566 -  88867:        17 
 70006 -  79485:        39 
 60191 -  69891:        51 
 50210 -  59643:        98 
 40106 -  49917:       191 
 30015 -  39986:       448 
 20006 -  29992:      1068 
 10000 -  19995:      4187 
  9001 -   9999:       942 
  8001 -   8998:      1202 
  7000 -   7999:      1489 
  6000 -   6999:      1927 
  5000 -   5999:      2379 
  4000 -   4999:      3266 
  3000 -   3999:      4580 
  2000 -   2999:      6979 
  1000 -   1999:      9654 
   900 -    999:      1176 
   800 -    899:      1346 
   700 -    799:      1658 
   600 -    699:      2405 
   500 -    599:      4742 
   400 -    499:     13047 
   300 -    399:     26578 
   200 -    299:    361389 
   100 -    199: 135260255 
    90 -     99:   7874207 
    80 -     89:   7147630 
    70 -     79:   5128367 
    63 -     69:   2427507

 138,219,089 out of 158,805,551 contain one of the frequent kmers

CGW

  • Monitor cgw
 ps -C cgw
 PID  PPID %MEM   RSZ %CPU STIME     TIME CMD
  8563  8560 95.2 251872528 88.2 13:24 01:47:56 /fs/szdevel/dpuiu/SourceForge/wgs-6.1/Linux-amd64/bin/cgw  ...

 top -b -p 8563 -d 10 | grep dpuiu > cgw.resource_usage.log
  • Failure 1:
 tail 7-0-CGW/cgw.out 
 ...
 Processed 158,288,858 unitigs with 326,296,236 fragments    #Bumble bee : Processed 61,930,044 unitigs with 301,738,113 fragments
 * Loaded dist s_2_3kb,1 (3000 +/- 300)
 * Loaded dist s_2_8kb,2 (8000 +/- 800)
 * Loaded dist s_3,3 (475 +/- 47.5)
 ...
 * Splitting chimeric input unitigs
 LIB 1 mu = 15.318100 sigma = 89.035478
 LIB 2 mu = 8000.000000 sigma = 800.000000
 LIB 3 mu = 337.817628 sigma = 26.699549
 ...
 minLength = 460
 minSplit  = -429
 Splitting unitig 47689 into as many as 3 unitigs at intervals:  22905,22906
 ..
 Splitting unitig 158234882 into as many as 3 unitigs at intervals: 124,136
 * BuildGraphEdgesDirectly

 Fix (partial): 
 add "-I" flag to cgw in runCA
 cat 7-0-CGW/cgw.out
 ...
 *** BuildGraphEdgesDirectly Operated on 171664374 fragments
  • Failure 2:
 tail 7-0-CGW/cgw.out
 **** Calling CheckEdgesAgainstOverlapper ****
 **** Survived CheckEdgesAgainstOverlapper with 0 failures****
 * Allocating Contig Graph with 158289029 nodes and 14055921 edges
 Could not calloc memory (25326244640 * 1 bytes = 25326244640)
 cgw: AS_UTL_alloc.C:55: void* safe_calloc(size_t, size_t): Assertion `p != __null' failed.

 Fix : delete single fragment unitigs (tigStore) and the fragments asseociated with them (gkpStore)

   158,288,858    unitigs total
   154,631,861    unitigs to delete (single frg unitigs)
     3,656,997    unitigs to keep

   326,236,387    frg total
   154,631,861    frg to delete     (single frg unitigs)
   171,604,526    frg to keep

Stats

  .                    elem         min    q1     q2     q3     max        mean       n50        sum            
  scf                  11,768       109    4232   8819   22525  741575**   21676      51546      255094767      
  ctg                  16,852       64     3501   7433   17418  317387**   15122      31217      254844680    
  ctg100+              16,822       100    3514   7453   17442  317387     15149      31217      254842109     
  deg                  3,388,183    64     125    138    168    4608       150        148        511490229      
  utg                  3,657,090    64     124    138    169    168543     216        179        792973816      
  
  totalReads           326,236,387
  usableReads          171,664,375
  singletonReads       1
  • Mate stats (%)
 lib  badLong  badOuttie  badSame  bothDegen  bothSurrogate  diffScaffold  good   notMated  oneDegen  oneSurrogate  
 1    0.01     53.9       0        15.57      0.36           0.24          0.4    18.33     9.45      1.68          
 2    0        1.1        0        26.09      0.31           0.09          0.03   61.57     9.42      1.32          
 3    0.02     0          0        7.94       0.5            0.11          69.91  19.88     1.03      0.48          
 4    0.02     0          0        7.78       0.48           0.11          69     21.01     1.01      0.47          
 5    0.02     0          0        7.71       0.47           0.11          69.02  21.08     1         0.47          
 6    0.02     0          0        7.72       0.48           0.11          69.76  20.32     1         0.47          
 7    0.02     0          0        7.74       0.48           0.11          69.2   20.89     1         0.46

 lib    mates     ULK      CLK      SLK   
 1      2494975   961180   521360   14    
 2      14560     14483    12375    4     
 3      13510875  2120862  1169185  2502  
 4      13100370  2048500  1128523  2392  
 5      12974843  2017970  1110081  2467  
 6      13304733  2059656  1125182  2481  
 7      12774596  1986181  1095046  2386

 cat SLK.asm | grep ^mea | sed 's/mea://' | getSummary.pl -t SLK | pretty -int -o
 cat CLK.asm | grep ^mea | sed 's/mea://' | getSummary.pl -t CLK | pretty -int -o
 cat ULK.asm | grep ^mea | sed 's/mea://' | getSummary.pl -t ULK | pretty -int -o
 cat SCF.asm | grep mea | grep -v mea:0.000 | sed 's/mea://' | getSummary.pl -t CTP | pretty -int -o

 .      elem     min      q1      q2    q3    max  mean   n50   sum       
 SLK    981      -53589   -484    -328  -183  4607 -1311  4607  -1286505  
 CLK    2714158  -488421  -42     34    73    7856 -682   7856  -1853032141  
 ULK    3576611  -188986  -71     24    69    7856 -338   7856  -1209800223  
 CTP    4987     -33482   -19     -6    15    10646 1     10646  8602

Location

 mulberry:/scratch2/dpuiu/Megachile_rotundata/Assembly/wgs-noOBT/  (to be deleted)
 /fs/szattic-asmg5/Bees/Megachile_rotundata/Assembly/wgs-noOBT/    **
  • Ftp
 ftp://ftp.cbcb.umd.edu/pub/data/assembly/Megachile_rotundata.wgs.1/
 /fs/ftp-cbcb/pub/data/assembly/Megachile_rotundata.wgs.1
  • Try:
    • bog
 -b         Break promisciuous unitigs at unitig intersection points              => delete
 -m 7       Break a unitig if a region has more than 7 bad mates                  => increase to 1000
  • cgw :
 -m <min>     Number of mate samples to recompute an insert size, default is 100 => increase to ?

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            
 scaf                 7863       102    903    3272   17692  2,338,728**  37825      240,706    297423517     # N50 for Bee was 1.17M
 contigs              9742349    31     32     33     37     114,832      60         44         585430821      
 contigs(>100bp)      177327     100    131    261    1398   114,832      1333       3897**     236496823     # N50 for Bee was 7K
  • Location
 /fs/szattic-asmg5/Bees/Megachile_rotundata/Assembly/assembly5kbForAll

SOAPdenovo (Daniela)

Stats

 .                    elem       min    q1     q2     q3     max          mean       n50        sum            
 scaff                25,119     351    1896   4444   10914  1,102,803    11041      26876      277,338,897
 scaff(all)           51,551     100    121    489    4320   1,102,830    5516       25940      284,368,749 

 scaffContigs(all)    263,977    33     78     149    878    121,523      914        3273       241,406,149  # <= scafffasta2fasta.pl

 contigs(all)         6,917,796  31     32     34     40     121,554      70         73         487,401,812      
 contigs(>100bp)      210,666    100    124    222    1174   121,554      1108       3138       233,563,401

 reads                340,437,486
 readsOnContigs       171,212,613

Alignments

  • Align reads to the scaffolds
 soap2-index asm.K31.scafSeq
 mkdir soap2-index
 mv asm.K31.scafSeq.index.* soap2-index/
   
 soap2 -D soap2-index/asm.K31.scafSeq.index  -a s_2_1_1.1kb_sequence.txt -b s_2_2_1.1kb_sequence.txt -l 32 -p 16 -v 2 -m 800  -x 1400  -o s_2_1.1kb.mated.soap2 -2 s_2_1.1kb.single.soap2 
 soap2 -D soap2-index/asm.K31.scafSeq.index  -a s_2_1_3kb_sequence.txt   -b s_2_2_3kb_sequence.txt   -l 32 -p 16 -v 2 -m 2000 -x 4000  -o s_2_3kb.mated.soap2   -2 s_2_3kb.single.soap2   -R 
 soap2 -D soap2-index/asm.K31.scafSeq.index  -a s_2_1_5kb_sequence.txt   -b s_2_2_5kb_sequence.txt   -l 32 -p 16 -v 2 -m 4000 -x 6000  -o s_2_5kb.mated.soap2   -2 s_2_5kb.single.soap2   -R 
 soap2 -D soap2-index/asm.K31.scafSeq.index  -a s_2_1_8kb_sequence.txt   -b s_2_2_8kb_sequence.txt   -l 32 -p 16 -v 2 -m 6000 -x 10000 -o s_2_8kb.mated.soap2   -2 s_2_8kb.single.soap2   -R
 soap2 -D soap2-index/asm.K31.scafSeq.index  -a s_3_1_sequence.txt       -b s_3_2_sequence.txt       -l 32 -p 16 -v 2 -m 200 -x  400   -o s_3.mated.soap2       -2 s_3.single.soap2 
 ...
                   mates           mated       single      single.diffScaff        single.sameScaff
 s_2_1.1kb         32,634,858      1,466,112   8,014,900   131,084                 585,668 

 s_2_3kb           21,563,283      1,545,114   8,449,321   341,974                 1,203,570
 s_2_3kb.trim64    21,563,283      4,291,750*  12,156,958  1,484,498*              3,457,492
 s_2_3kb.filter    4,823,235       3,730       3,618,426   38,562                  3,017,172

 s_2_5kb           36,218,589      5,639,332   44,533,553  4,784,348               30,621,038

 s_2_8kb           198,377         1,068       32,280      1,168                   2,842
 s_2_8kb.trim64    198,377         5,508*      48,877      4,258*                  3,532
 s_2_8kb.filter    111,267         20          33,819      372                     27,300  

 s_3               35,548,153      15,521,020  4,589,276   37,564                  651,924

Location

 mulberry:/scratch2/dpuiu/Megachile_rotundata/Assembly/SOAPdenovo/

SOAPdenovo ; partial : s_[34567] ; no repeats **

  • Slightly better results than we got when the s_2_?kb libs were used

Stats

  .                 elem       min    q1     q2     q3     max         mean       n50        sum            
  scaff             24,602     333    1724   4380   11049  1,103,462   11135      27887      273963709
  scaff(all)        40,883     100    146    1366   5960   1,103,447   6833       27088      279355387

  scaffContigs(all) 231,559    33     75     153    982    148,167     1039       3950       240752397
 
  contigs(all)      2,515,516  31     33     36     52     148,198     131        1880       330512911      
  contigs(100bp+)   184,395    100    127    235    1316   148,198**   1263       3730       232932308

Location

ginkgo: /scratch1/dpuiu/Megachile_rotundata/Assembly/SOAPdenovo-partial.3.noRepeats/

Aligning long inserts to this assembly =

  • Should trim the linker (mostly at the beginning of the reads )
 show-coords -H 1000_12.filter-1.delta | sort -k19 | p '$F[18]=~s/[12]$//; print $p,$_,"\n" if($P[18] eq $F[18] and $P[17] ne $F[17]); $p=$_; @P=@F;' | pretty
 ...
 cat *-1000*filter*delta | show-coords.pl | sort -k19 | ...

 417      469    |  2   54   |  53  53    |  98.11  |  2148   54   |  2.47   98.15  |  scaffold16793  HWI-EAS385_0062:2:1:10306:11665#CAGATC/1  [CONTAINS]  
 10634    10719  |  39  124  |  86  86    |  96.51  |  10732  124  |  0.8    69.35  |  scaffold21864  HWI-EAS385_0062:2:1:10306:11665#CAGATC/2  .           
 1        38     |  40  3    |  38  38    |  100    |  42     124  |  90.48  30.65  |  linker.rev     HWI-EAS385_0062:2:1:10306:11665#CAGATC/2  .           

 1       38      |  40  3    |  38   38   |  100    |  42    124   |  90.48  30.65  |  linker.fwd    HWI-EAS385_0062:2:1:10759:13923#CAGATC/1  .           
 1       82      |  41  122  |  82   82   |  95.12  |  7015  124   |  1.17   66.13  |  scaffold5186  HWI-EAS385_0062:2:1:10759:13923#CAGATC/1  .           
 5293    5416    |  1   124  |  124  124  |  94.35  |  7427  124   |  1.67   100    |  scaffold7361  HWI-EAS385_0062:2:1:10759:13923#CAGATC/2  [CONTAINS]

SOAPdenovo ; s_2_3kb & s_ 2_8kb soap-aligned & trim64; s_2_1.1k & s_2_1.4k ; s_[34567] no repeat **

Stats

  .                     elem       min    q1     q2     q3     max         mean       n50        sum            
  scaf                  6242       228    640    2061   10681  3,022,211   42964      456,467    268,183,797      
  scafSeq               17702      100    119    178    830    2,999,853   15136      452,601    267,954,678      
 
  contigs               4138734    32     32     34     41     111,437     94         1286       389,303,351      
  contigs100+           181683     100    131    252    1385   111,437     1307       3789       237,563,819

  scafSeqContigs        124749     2      90     466    2237   123,623     1970       5,800      245,850,885 
  scafSeqContigsClosed  25288      2      130    382    5576   626,941     10218      61,363     258,400,185   # after running GapCloser
  • GC contig cvg bias
Megachile.contig10000.png

Scaffold links

 perl ~/bin/remapSOAPscafId.pl *.scaf -p 5 | p 'print $_ unless($F[-2]=~/DOWN/ and $F[-1]=~/UP/);' | more
 ...
>2 76 50030
 2.2     206     58      #DOWN   #UP     543.152:5:1248
 2.4     611     1458    #DOWN   #UP     543.152:26:1477
 2.24    18951   911     #DOWN   2268.26:31:139  #UP
 2.25    20219   2382    #DOWN   #UP     2268.26:28:212
 2.40    34053   150     #DOWN   1363.46:10:1041 #UP
 2.42    34434   76      #DOWN   1363.46:6:804   #UP
 2.43    34500   70      #DOWN   1363.46:6:682   #UP
 2.60    41566   200     #DOWN   808.70:8:219    #UP
 2.62    42030   1405    #DOWN   #UP     808.70:9:82
 ..
 >543 153 191782
 543.63  108072  1765    #DOWN   158.305:5:198   #UP
 543.66  110202  77      #DOWN   #UP     158.305:6:185
 543.98  163144  810     #DOWN   1285.8:25:206   #UP
 543.99  164653  393     #DOWN   #UP     91.485:6:197
 543.121 176938  49      #DOWN   #UP     985.12:24:88
 543.125 178897  102     #DOWN   234.161:5:275   #UP
 543.128 179479  63      #DOWN   220.220:11:214  1951.10:14:284  576.114:16:256  #UP     576.116:8:474   234.161:8:178
 543.152 188359  3153    #DOWN   2.4:26:1477     2.2:5:1248      #UP
 ...

SOAPdenovo vs CA

Aligns contigs 200+bp with nucmer.amos

 set REFN=`grep -c ">" ref.seq`
 @ REFN/=20
 nucmer.amos -D REF=ref -D QRY=qry -D REFN=$REFN ref-qry

 .                  elem       min    q1     q2     q3     max        mean       n50        sum            
 ref(CA)            16507      200    3678   7660   17785  317387     15436      31240      254794314      
 qry(SOAPdenovo)    100877     200    499    1224   2328   111437     2248       4103       226807646      



 cat ref-qry.delta | grep "^>" | sed 's/>//' | awk '{print $1,$3}' | sort -u  | getSummary.pl -i 1
 .                   elem       min    q1     q2     q3     max        mean       n50        sum            
 ref-hits            16195      200    3816   7848   18116  317387     15711      31309      254444709      
 qry-hits            95343      200    610    1307   2427   111437     2356       4171       224616404      

 .                   elem       min    q1     q2     q3     max        mean       n50        sum            
 ref-miss            312        200    244    483    1575   13984      1121       2262       349605         
 qry-miss            5534       200    235    298    442    5658       396        421        2191242

Location

mulberry: /scratch2/dpuiu/Megachile_rotundata/Assembly/SOAPdenovo.noRepeats.trim64/ (to be deleted)
/fs/szattic-asmg5/Bees/Megachile_rotundata/Assembly/SOAPdenovo.noRepeats.trim64/ **
/fs/ftp-cbcb/pub/data/assembly/Megachile_rotundata.SOAPdenovo.2/ =>  ftp://ftp.cbcb.umd.edu/pub/data/assembly/Megachile_rotundata.SOAPdenovo.2/

SOAPdenovo ; s_2_3kb & s_ 2_8kb soap-aligned & trim64; s_2_1.1k,s_2_1.4k,s_[34567] no repeat & trim64

  • Trimming all reads to the first 64bp does not change the results much (actually getting a little worse)

Stats

 .                     elem       min    q1     q2     q3     max         mean       n50        sum           
 scaf                  7615       441    896    2238   11462  2394735     37960      326734     289063269 
 scafSeq               23906      100    118    170    914    2396948     12271      324184     293340377

 contigs               1738246    32     33     37     61     122972      174        2037       302039034      
 contigs100+           214540     100    135    264    1263   122972      1110       2941       238063650

SOAPdenovo K=31 (new data)

  • Location
 /scratch1/dpuiu/Megachile_rotundata/Assembly/SOAPdenovo/K31
 ftp://ftp.cbcb.umd.edu/pub/data/assembly/Megachile_rotundata.SOAPdenovo.2011-03/
  • Assembly stats:
 .                 elem       min    q1     q2     q3     max        mean       n50        sum
 scf               16115      100    111    134    264    4173260    17307      1288790    278898652
 scfLen            16115      100    111    134    208    3855147    14496      950327     233605903 
 ctg               8829648    32     32     34     39     121554     62         3201       553630969

 scf2              16115      100    111    134    260    4033560    16539      1067152    266528571
 scf2Len           16115      100    111    134    259    4004254    16236      1071079    261641263
 ctg2              26490      3      120    243    5253   520023     9877       64739      261641355

 cat asm.K31.peGrads | tail -6 | p 'print $F[0], " ", $F[1]-$P[1],"\n"; @P=@F' | pretty
 350     330665408  
 1100    54707484   
 1400    86601708   
 3000    22148926   
 5300    37909358   
 8000    30203916   

 cat asm.K31.links | awk '{print $5}' | uniq -c  | awk '{print $2,$1}'
 350     7375561
 1100    579996
 1400    604951
 3000    340192
 5300    669339
 8000    184868

 7375561 350
  579996 1100
  604951 1400
  340192 3000
  669339 5300
  184868 8000

SOAPdenovo K=47 (new data) **

  .                    elem       min    q1     q2     q3     max        mean       n50        sum           
  scaf                 3495       259    408    757    4763   6,173,378  82382      2,124,089  287,925,734
  scafSeq              31774      100    110    128    172    6,174,792  9215       2,124,853* 292,784,153 
  scafSeq2             31774      100    110    128    172    5,876,085  8689       1,814,396  276,082,351
  
  contigs              10217806   48     48     50     56     175,671     77        4,701      792,925,336     
  contigs100+          224315     100    126    177    933    175,671     1134      4,701      254,309,160 
  scafSeqContigsClosed 43232      2      114    147    740    479,105     6228      63,194*    269,243,097

Location: 

 /fs/szattic-asmg5/Bees/Megachile_rotundata/Assembly/SOAPdenovo.10libs.K47

SOAPdenovo vs CA

             elem       min    q1     q2     q3     max        mean       n50        sum 
 CA          16848      64     3501   7433   17428  317387     15124      31240      254808119      
 SO          43232      2      114    147    740    479105     6228       63194      269243097      

 CA.no_hits  315        64     124    150    188    1353       168        167        52986          
 SO.no_hits  29679      2      109    123    151    5766       185        158        5489188

Allpaths-LG (experiment)

  • Shred SOAPdenovo K=47 contigs >=180bp ; use them as fragment library
 .                elem    min  q1    q2    q3     max      mean   n50     sum        
 contig.180+      110580  180  264   964   2321   175671   2166   4701    239535581
  • Libraries used: (in_libs.csv)
 library_name,    project_name,  organism_name,  type,      paired,  frag_size,  frag_stddev,  insert_size,  insert_stddev,  read_orientation,  genomic_start,  genomic_end   #mates
 frag,            genome,        genome,         fragment,  1,       180,        20,           ,             ,               inward,            0,              0             22,024,446  # originally 176,833,196 fragments insert_size=475bp
 s_2_1.1kb,       genome,        genome,         jumping,   1,       ,           ,             1100,         110,            inward,            0,              0             32,634,858
 s_2_1.4kb,       genome,        genome,         jumping,   1,       ,           ,             1400,         140,            inward,            0,              0             50,861,645
 s_7_5kb,         genome,        genome,         jumping,   1,       ,           ,             5300,         530,            outward,           0,              0             29,111,787
 s_7_8kb,         genome,        genome,         jumping,   1,       ,           ,             8000,         800,            outward,           1,              37            25,328,718

 type             #mates.original  #mates.corrected
 frag             22,024,446       21,925,564
 jumping          137,937,008      5,465,197
  • Assembly stats:
  .                    elem       min    q1     q2     q3     max        mean       n50        sum           
  scf                  1964       1      1187   1550   6679   6,117,056  128,398    1,806,225  252,173,219
  ctg                  36959      1      1836   3140   6343   278,684    6,061      8,784      224,007,758

Genbank submission

  • Information
 Project Type: Single Species Project  
 Contacts: Daniela Puiu dpuiu@umiacs.umd.edu, Steven Salzberg salzberg@umiacs.umd.edu
 Submitting Organization: University of Illinois & University of Maryland
 Sequencing Center: Keck Center for Comparative and Functional Genomics, University of Illinois  BCUI  Biotechnology Center, Univ. Illinois (BCUI)
 Consortium Name: Alfalfa Leafcutter Bee Genome Consortium
 Organism Name: Megachile rotundata
 Strain/isolate/breed: North American commercial strain
 Locus Tag Prefix: MROT #3+ letters
 Source of DNA used for sequencing: whole body, haploid brother males
 Sequencing Method: wgs  
 Sequencing Technology: Illumina
 Estimated Genome Size: 250Mb # the haploid genome size
 Brief description of the importance: Megachile rotundata, alfalfa leafcutting bee, is a solitary bee species. It is the #3 agricultural pollinator in the United States and is commercially managed for alfalfa seed production.
 Comments to the staff:
  DNA
  whole genome sequencing
  single genome;
  no annotation
  assembly name MROT_1.0
  assembly method: SOAPdenovo Assembler v_1.05
  plan to update: ?
  expect to release : ?
  strain information to be submitted soon 
  genome coverage: 300x
  sequencing technology:  Illumina GA IIx  ??
  Author list: 
         Gene E. Robinson,    
         Hugh M. Robertson,   
         Matthew E. Hudson,   
         Kim Walden,          
         Brielle J. Fischman, 
         Theresa Pitts-Singer,  
         Rosalind James
         Steven Salzberg,     
         Daniela Puiu,        
         Tanja Magoc,         
         David Kelley,        
         Aleksey Zimin ,
  • Best assembly : SOAPdenovo K=47
  /fs/szattic-asmg5/Bees/Megachile_rotundata/Assembly/SOAPdenovo.10libs.K47/genbank[1-3]
  • Sequence length statistics [1]:
                      number     min    max        mean       n50        sum           
 scaffolds            31774      100    5876085    8689       1814396    276082351     
 contigs              42781      100    479105     6293       69010      269224383
  • Removed 6 contaminants & scaff < 200bp [2]
 .                    elem       min    max        mean       n50        sum            
 scaffolds            6367       200    5876085    42857      1699680    272873468    
 contigs              17374      100    479105     15311      64153      266015500
  • NCBI comments: There are 668 contigs that are <200bp remaining. Some of these are internal components of scaffolds, but many of these are at the ends of scaffolds so should be removed. For example, 249 of them are the first or component of a scaffold or the only component of a singleton scaffold (list.ShortTerminalContigs). And some of them are the only components of a multi-component scaffold, eg these two scaffolds are made entirely of short contigs ... 
 .                    elem       min    max        mean       n50        sum            
 scaffolds            6266       200    5876085    43514      1814396    272660569      # 101 scaffolds deleted
 contigs              16706      200    479105     15917      69010      265916502      # 668 contigs deleted
  • GPID Organism name Accession
 -----------------------------------------------
 66515	Megachile rotundata	AFJA00000000

Please cite the accession number as usual:

   This Whole Genome Shotgun project has been deposited at 
   DDBJ/EMBL/GenBank under the accession AFJA00000000.
   The version described in this paper is the first version,
   AFJA01000000.
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