Bumblebee: Difference between revisions

From Cbcb
Jump to navigation Jump to search
Line 114: Line 114:


== D Kelly's trimming ==
== 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                    301738113  .    .    .    .    .    .      .    31958664367 
 
* Mates
  s_1.mates                3152991   
  s_2.mates                768603   
  s_3.mates                9633768   
  s_5.mates                10675551 
  s_6.mates                9954243   
  s_7.mates                10262620 
  s_8.mates                3513074   
  total                    47960850


= Assembly =
= Assembly =

Revision as of 19:18, 30 March 2010

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       Coverage    Comments
 1      3K(2..6,avg 4K)   124        34,944,099   14X         865,687(1.2%) reads have qual==0
 2      8K(7..9,avg 8K)   124        32,540,640   13X

 3      400               124        34,745,750               # gDNA ; originally thought to be 500bp insert instead of 400
 5      400                          34,601,239
 6      400                          34,553,857
 7      400                          34,682,612
 8      400                          12,975,839
  
 3-5                                 138,583,458  69X
  • Adaptors
 >circularizarion
 CGTAATAACTTCGTATAGCATACATTATACGAAGTTATACGA
 >circularizarion.revcomp
 TCGTATAACTTCGTATAATGTATGCTATACGAAGTTATTACG 
 >5
 GATCGGAAGAGCGGTTCAGCAGGAATGCCGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG
 >3
 CGGCATTCCTGCTGAACCGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT

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)
  • Location:
 /fs/szattic-asmg4/Bees/Bombus_impatiens/
 /fs/szattic-asmg4/Bees/Bombus_impatiens/frg/   # frg files
 ginkgo:/scratch1/Bombus_impatiens/Data/

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                     301738113  .    .    .    .    .    .       .    31958664367
  • Mates
 s_1.mates                 3152991    
 s_2.mates                 768603     
 s_3.mates                 9633768    
 s_5.mates                 10675551   
 s_6.mates                 9954243    
 s_7.mates                 10262620   
 s_8.mates                 3513074    
 total                     47960850

Assembly

CA.bog

  • 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 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
Retrieved from "https://wiki.umiacs.umd.edu/cbcb/index.php?title=Bumblebee&oldid=6957"