Bumblebee

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Data

  • ~ 500B genome
  • Complete mitochondrion genomes:
 NC_011923.1    15468  14.67  Bombus hypocrita sapporoensis mitochondrion, complete genome
 NC_010967.1    16434  13.22  Bombus ignitus mitochondrion, complete genome
 only 88% identity; no rearrangements, only snps, short indels

Traces

  • 7 pairs of data files (paired ends) : lanes 1..3,5..8 (lane 4 wasn't used)
 Lane   Insert            ReadLen    #Mates         #Reads         Coverage   Orientation     Comments
 1      3K(2..6,avg 4K)   124        34,944,099                    14X        outie           865,687(1.2%) reads have qual==0
 2      8K(7..9,avg 8K)   124        32,540,640                    13X        outie

 3      400               124        34,745,750                    .                           gDNA ; originally thought to be 500bp insert instead of 400
 5      400               124        34,601,239                    . 
 6      400               124        34,553,857                    . 
 7      400               124        34,682,612                    . 
 8      400               124        12,975,839                    . 
  
 3-8                                 151,559,297     303,118,594   75X
 1-8                                 219,044,036     438,088,072   108X

 Two new 3kb libraries to come; ~ 70% are true pair ends
  • Adaptors
 >circularizarion
 CGTAATAACTTCGTATAGCATACATTATACGAAGTTATACGA
 >circularizarion.revcomp
 TCGTATAACTTCGTATAATGTATGCTATACGAAGTTATTACG 
 >5
 GATCGGAAGAGCGGTTCAGCAGGAATGCCGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG
 >3
 CGGCATTCCTGCTGAACCGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT
  • Location
 /fs/szattic-asmg4/Bees/Bombus_impatiens/s_[1235678]_[12]_sequence.txt : s_1-8 libs
 /fs/szattic-asmg4/Bees/Bombus_impatiens/3kb_Bimpatiens.txt            : 88 reads

Tasks to figure out

  1. Erroneous reads/bases, which we need to correct or discard
  2. GC bias, so we can compute a-stats properly
  3. Redundancy in the long paired ends, which are lane 1 and lane 2.
  • Used the 454 protocol to circularize the DNA for sequencing with the Illumina instrument.
    • Some reads will begin in the circularization adaptor and thus will have only one usable read
    • Some reads have a few bases of DNA sequence and hit the circularization adaptor right away
    • Most reads will have at least 36bp from each end before hitting the adaptor.
    • Many reads will not have any adaptor to trim (>125bp of DNA sequence at both ends of the adaptor)
  • A small but significant number of reads from the 3kb and 8kb libraries are not recircularized.
    • Thus their mate distance is +400bp rather than -3kb or -8kb.
    • It's apparently the result of a faulty batch of cre recombinase. This causes problems with contiging and scaffolding.
    • It is possible to remove these reads by removing
  1. mate pairs where neither read is trimmed (thus no adapter ligation may have occurred)
  2. mate pairs where one read begins with the adapter sequence.
    • We are working on this, up to now our paired assembly stages have been disappointing.
  • Matt's idea is to exclude all mate pair reads that don't have evidence of the linker with flanking useful sequence, as a way to avoid uncircularized molecules that will give misleading "mate pairs" only 400 bp apart.
    • There has been no trimming of the adaptor, which is the 42 base 454 adaptor, so its presence can be used to indicate potentially good mate pairs.
    • Even tossing half the mate pairs might not be a problem, as we have perhaps too many anyway.
    • But you will also need to toss redundant mate pairs, and that will indeed reduce the total a lot.
    • Just to be clear - the 500 base mate pairs should have no such problems, except that as Matt has found from his preliminary assembly, the mean fragment length is actually 400 bp rather than 500 bp (and the 3 and 8 kb PE reads are typically shorter than nominally given, e.g. more like 2.5 and 6 kb).
    • You'll also need to throw out all the reads where one of the mates /starts/ with linker, I assume you'd do that anyway. We're also working on ways round this; one might be when we get a better assembly we'll find some better characteristic to filter the unrecircularized reads on.

Trimming

  • Quality trimming: trim all bases with fastq quality eq "B" (0)
  cat *fastq | ~/bin/fastq2clq.pl
  • Adaptor trimming: Align all subsets to adaptors
 C CGTAATAACTTCGTATAGCATACATTATACGAAGTTATACGA
 3 CGGCATTCCTGCTGAACCGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT
 5 GATCGGAAGAGCGGTTCAGCAGGAATGCCGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG

 id  len    gc%
 C   42     30.95  
 3   52     55.77  
 5   67     59.70  

 nucmer -l 8 -c 16 -b 32 -g 32 adaptors.seq  ...
 delta-filter -l 16 -q   ...
 cat *.filter-q.delta | ~/bin/delta2clr53.pl -5 5,3 ...
  • Adaptor/primers median positions in the reads
 Lib       adaptorPos     5'primerPos 3'primerPos 
 s_1       34-75          0-36        0-19
 s_2       2-40           0-36        0-19
  • Mate stats
 Lib        #mates    adaptor.bothMates   adaptor.noMate   adaptor.oneMate   adaptor.oneMate(filtered)   
 s_1        34944099  269156              9804247          24870696          6048164(17.3%)               
 s_2        32540640  91528               16181288         16267824          1061858( 3.2%)   
 total      67484739                                                         7110022(10.5%)
  • Filtered read clr stats
 Lib        #mates     #reads       min    q1     q2     q3     max        mean       n50        sum            
 s_1        6048164    12096328     64     80     95     115    124        96.56      101        1,168,064,632     
 s_2        1061858    2123716      64     80     96     115    124        96.59      101          205,122,226 
 total      7110022    14220044     64     80     95     115    124        96.57      101        1,373,186,858
  • Filtered read GC% stats
 Lib        #reads       min    q1     q2     q3     max        mean       n50                    
 s_1        12096322     0.00   31.93  36.26  42.35  100.00     37.45      38        
 s_2        2123715      0.00   31.88  36.46  42.48  100.00     37.56      38
  • Other frequent kmers
 26mer : ACGTTATAACGTATTACGTTATATGG -> revcomp -> CCATATAACGTAATACGTTATAACGT : ~10% of the traces
 10mer : AAAAAAAAAA                               TTTTTTTTTT                 : ~32% of the traces    
 53mer: CGATTTCCATGGCGTCGTTTGAGGATTCCAATACGGCGAACCTGTTGTGAGTG                : ~2% of the mito seqs (either begin or end); not present in the 2 complete mito's (probably ok)
  • Data processing pipeline
 s_[12]    : /nfshomes/dpuiu/bin/fastq2adaptorFreeFrg.amos
 s_[35678] : /nfshomes/dpuiu/bin/fastqfrg.amos
  • Location:
 /fs/szattic-asmg4/Bees/Bombus_impatiens/s_[1235678]_[12]_sequence.txt                   # original fastq files
 /fs/szattic-asmg4/Bees/Bombus_impatiens/error_free/s_[1235678]_[12]_sequence.cor.txt    # corrected fastq files

 /fs/szattic-asmg5/Bees/Bombus_impatiens/s_[12]/all/s_[12].64.rev.frg                    # adaptor free s_[12] mated reads
 /fs/szattic-asmg5/Bees/Bombus_impatiens/error_free/frg/s_[1235678].frg                  # adaptor free & corrected s_[1235678] reads

D Kelly's trimming

  • Reads
 .                         elem       min  q1   q2   q3   max  mean    n50  sum          
 s_1_1_sequence.cor.txt    5436814    30   71   90   115  124  89.70   100  487673256    
 s_1_2_sequence.cor.txt    5864225    30   77   92   110  124  93.27   98   546956864    
 s_2_1_sequence.cor.txt    1053858    30   80   96   118  124  97.16   102  102395785    
 s_2_2_sequence.cor.txt    1041846    30   78   93   110  124  93.51   99   97426679     
 s_3_1_sequence.cor.txt    33668302   30   105  124  124  124  111.04  124  3738530761   
 s_3_2_sequence.cor.txt    32554322   30   82   108  124  124  100.42  120  3269039697   
 s_5_1_sequence.cor.txt    33579744   30   109  124  124  124  111.65  124  3749192427   
 s_5_2_sequence.cor.txt    32465412   30   84   112  124  124  102.13  124  3315553171   
 s_6_1_sequence.cor.txt    33535725   30   109  124  124  124  111.60  124  3742602285   
 s_6_2_sequence.cor.txt    32390877   30   83   109  124  124  100.92  123  3268875471   
 s_7_1_sequence.cor.txt    33674235   30   112  124  124  124  112.19  124  3777917379   
 s_7_2_sequence.cor.txt    32518568   30   84   110  124  124  101.60  124  3303807321   
 s_8_1_sequence.cor.txt    11777821   30   113  124  124  124  112.30  124  1322658923   
 s_8_2_sequence.cor.txt    12176364   30   84   110  124  124  101.51  124  1236034348   
 total                     301,738,113  .  .    .    .    .    .       .    31958664367  
 total (64bp+)             275,763,594  .  .    .    .    .    .       .    30693543885
  • Mates
 s_1.mates                 5320163    
 s_2.mates                 1034086    
 s_3.mates                 31857304   
 s_5.mates                 31820553   
 s_6.mates                 31748713   
 s_7.mates                 31891468   
 s_8.mates                 11168258   
 total                     144,840,545
  • Files
 /fs/szattic-asmg4/Bees/Bombus_impatiens/error_free/frg/s_?.frg                    # frg 
 /fs/szattic-asmg4/Bees/Bombus_impatiens/error_free/s_?_[12]_sequence.cor.txt      # fastq

Assembly

CA.bog s_1

  • Input  : 31*2M s_1 reads trimmed to 100bp & formatted by fastqToCA
  • Spec file
 unitigger =                      bog
 ovlOverlapper =                  mer
 obtMerThreshold =                400
 ovlMerThreshold =                120
 doOverlapTrimming =              0
  • Unitigger : max utg len=852bp
  • Consensus after unitigger : 3 out of 129 jobs failed
  • Location
 /scratch1/Bombus_impatiens/Assembly/31M

CA.utg s_1,2 filtered

  • Input: 7*2M s_1 & s_2 reads formatted by convert-fasta-to-v2.pl
  • Filter:
    • only one read from the mate contains the adaptor in the 64..124bp range
    • both reads from the mate have good quality in the 0..64bp range
  • Spec file
 unitigger            =  utg  
 ovlOverlapper        =  ovl  
 obtMerThreshold      =  400
 ovlMerThreshold      =  200
 doOverlapTrimming    =  0     => reads < 64 bp atre trimmed by the gatekeeper; no need to trim
  • ovlStore stats
 #ovls/read
 reads      min    q1     q2     q3     max        mean       n50        sum            
 14220044   0      1      5      31     514        31.30      131        445090932
  • final stats
 .                    elem       min    q1     q2     q3     max        mean       n50        sum            
 ctg                  2          1004   1004   1055   1055   1055       1029.50    1055       2059           
 deg                  1433143    64     115    129    164    1028       145.79     145        208938949   
 assembled            7557472
 singletons           6662572
  • Mate redundancy
 cat 9-terminator/asm.posmap.frgdeg  | sort -nk2 -nk3 | perl ~/bin/posmap2ovl.pl | p 'chop $F[1]; chop $F[2]; print "$F[1] $F[2]\n"; print "$F[2] $F[1]\n";' | count.pl -m 2 | more
  • Location
 /scratch1/Bombus_impatiens/Assembly/14M.redo

SOAPdenovo s_3..8 orig (CBCB)

  • Kmer=23
  • Input: s_3,5,6,7,8 original reads
  • Error correction: no
  • Summary
 .                    elem       min    q1     q2     q3     max        mean       n50        sum            
 scf                  571,114    100    106    115    141    156066     162.19     137        92,628,126       
 ctg                  54049561   24     24     28     42     2049       35.04      35         1,893,660,606     
 gapSeq               49518      28     123    143    181    424        152.97     158        7,574,733

SOAPdenovo s_3..8 corr (CBCB)

  • Kmer=31
  • Input: s_3,5,6,7,8 reads
  • Error correction: yes
  • Summary
                    elem       min    q1     q2     q3     max        mean       n50        sum            
 scf                106,217    100    140    434    2418   102317     2,177      7099       23,1261,742

SOAPdenovo s_3..8 corr (Illinois)

  • Kmer=31
  • Input: s_3,5,6,7,8 reads
  • Error correction: yes; UIUC method
  • Summary
 .                    elem       min    q1     q2     q3     max        mean       n50        sum            
 scf                  17,550                                            13,460                236,225,169
 ctg                  4004102    31                          97,618     1916       6020       230,333,709
 ctg100bp+            120167     100

SOAPdenovo s_1..8 corr (Illinois)

  • Kmer=31
  • Input: s_1,2,3,5,6,7,8 reads
  • Error correction: yes; CBCB method
  • Summary
 .                    elem       min    q1     q2     q3     max        mean       n50        sum   
 ctg200bp+            43603      201    752    2786   9518   167534     7637.63    19895      333,023,446
  • Locations
 ftp://stan.cropsci.uiuc.edu/download/bombus1-k64-contigs.fa.min200
 ginkgo:/scratch1/Bombus_impatiens/Assembly/SOAPdenovo.s_1-8.cor.UIUC/

CA s_1..8 corr OBT (CBCB)

  • CA spec
 doOverlapTrimming =        1
 ovlOverlapper =            ovl
 unitigger =                bog 

 *BatchSize =               2000000
 *BlockSize =               2000000

 ovlMemory   =              8GB
  • Gatekeeper
 cat /scratch1/Bombus_impatiens/Assembly/CA.s_1-8.cor/asm.gkpStore.info | pretty -o
 LOAD                  STATS         
 
 7                     libInput      
 7                     libLoaded     
 0                     libErrors     
 
 301738113             frgInput      
 301738113             frgLoaded     
 25974519              frgErrors     
 
 144840545             lkgInput      
 124575590             lkgLoaded     
 20264955              lkgErrors     
 0                     lkgWarnings   
 
 275763594             numRandom     
 301738113             numNormal     
 
 
 GLOBAL                STATS         
 
 301738113             FRG           
 7                     LIB           
 
 LibraryName           numActiveFRG  numDeletedFRG  numMatedFRG  readLength   clearLength  
 GLOBAL                274282535     27455578       247151634    30524746650  29445986650  
 LegacyUnmatedReads    0             0              0            0            0            
 
 s_1                   10409320      891719         9039810      996306280    994802849    
 s_2                   2054575       41129          1987728      197829588    197593453    
 s_3                   59894531      6328093        53925476     2382786529   2127257586   
 s_5                   60127274      5917882        54512448     2459696212   2209078913   
 s_6                   59813485      6113117        54062058     2396359740   2152656620   
 s_7                   60278536      5914267        54647258     2476326572   2237423989   
 s_8                   21704814      2249371        18976856     2435572545   2347304056
  • Meryl
 meryl -Dh -s 0-mercounts/asm-C-ms22-cm0 
 Found 23686053415 mers.
 Found 265670791 distinct mers.
 Found 8682767 unique mers.
 ...
 #freq   count   %
 1       8682767 0.0327  0.0004
 2       4288602 0.0488  0.0007
 72*     3803015 0.7150  0.3229
 73      3797615 0.7293  0.3346
  • OBT/Overlaps jobs
 n=((301738113/2000000)+0.5)=151
 OBT/OVL jobs = n*n/2 =151*76=11476
 ~ 20 min/OBT job =~ 0.33 hr/OBT job  => 0.33*11476/32=5 days
  • OBT trimming
 key             sum             count           mean
 CLR             31958664367     301738113       105.915239043733
 OBTINITIAL      30779687358     301610679       102.051052900551
 OBTMERGE        30052401719     301610679       99.6397137483318
 LATEST          30052138325     301610679       99.638840456972


  • Failures:
    • overlapStore: too many files (max 10240) ; fix: recompile the code
    • chimera: FAILED file seems to be truncated but contains records from all libs; fix: link asm.chimera.report.FAILED asm.chimera.report
  • Location:
 ginkgo:/scratch1/Bombus_impatiens/Assembly/CA.s_1-8.cor

CA s_1..8 corr noOBT (CBCB)

  • CA spec
 doOverlapTrimming =              0
 ovlOverlapper =                  ovl
 unitigger =                      utg
  • Location:
 walnut:/scratch1/Bombus_impatiens/Assembly/CA.s_1-8.cor.noOBT
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