Chris:LabNotes/Microbiome/2011/2011-5-2

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Slide Shortening Assembly Test[edit]

Background[edit]

  • Sam tried to increase SAG contig length by using recruited sequences from metagenomic contigs (http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2011-4-19)
    • But results showed some decreased contig lengths after recruitment (ID2, ID9, ID11)
    • Dr. Zhang suggested that the decreased contig lengths may be fixed by slide shortening of contigs
  • On 4/29, I wrote perl script that could help perform the slide shortening of contigs
  • Here, I test the script/method of slide shortening to increase the contig length after recruitment
  • SAG sequence: ID9 because showed the lower contig length but showed relatively good contig length
    • Sequences found in: </media/disk-1/samchiang/Sub_tests_folder_imp/2_Extending_Mouse_Bac_SAG_contigs/BLAT_Meta2SAG_040311/Step4_pooled_together/SAG_A_ID9>
    • Test found in: </media/disk-2/cjwei/slide-shortening_Assembly_Test>
    • Used various sliding cut lengths:
      • Test 1: >1000bp contigs cut; step size = 200bp
      • Test 2: >2000bp contigs cut; step size = 100bp (same as "Whole Genome Amplification and Denovo Assembly of Single Bacterial Cells")
      • Test 3: >1000bp contigs cut; step size = 100bp
      • Test 4: >100bp contigs cut; step size = 10bp (most highly cut option)
      • Test 5: No sliding cut but have assembly
      • Test 6: Control (no sliding cut, no assembly -> just run abyss-fac on the combined file)
      • Test 7: SAG contigs (for comparison to determine if recruitment helped)
  • Also tried out the sliding cut on another assembly (see if can improve the average recruited assembly results) = ID1
    • Test 1: >2000bp contigs cut; step size = 100bp (was shown with ID9 that this produced highest N50 and highest sum)
    • Test 2: No sliding cut but have assembly
    • Test 3: Control (no sliding cut, no assembly -> just run abyss-fact on the combined file)
    • Test 4: SAG contigs (for comparison to determine if recruitment helped)

Procedure[edit]

  • Use Sliding Cut for combined SAG and recruited metagenome contigs:
perl contig_sliding_cut.pl SAG_A_ID9_and_recruted_Meta_all >SAG_A_ID9_and_recruted_Meta_all_slide_cut
  • Run Velveth
    • k-mer 27, -short
/media/disk-2/cjwei/Velvet/velveth SAG_A_ID9_and_recruted_Meta_all_slide_cut_Assembly/ 27 -fasta -short SAG_A_ID9_and_recruted_Meta_all_slide_cut
  • Run Velvetg
    • fixed setting as cov_cutoff 2, min-contig lgth 100
/media/disk-2/cjwei/Velvet/velvetg SAG_A_ID9_and_recruted_Meta_all_slide_cut_Assembly/ -cov_cutoff 2 -min_contig_lgth 100

Results[edit]

ID9 (problem assembly)[edit]

n       n:100   n:N50  min    median mean   N50    max    sum
7871    7871    737    100    196    465    1000   20981  3662057 contigs_1000_200.fa
7819    7819    710    100    197    473    1057   21081  3703868 contigs_2000_100.fa
7861    7861    726    100    196    469    1039   21081  3689257 contigs_1000_100.fa
7869    7869    718    100    196    468    1041   21091  3690448 contigs_100_10.fa
990     990     101    100    211    505    980    19794  500631  SAG_A_ID9_and_recruted_Meta_all_Assembly 
14828   14828   1140   100    170    403    902    38317  5988190 ../SAG_A_ID9_and_recruted_Meta_all
392     4392    490    100    187    400    769    9214   1758616 s6-8_A_ID9.contig.100up
  • Based on results, notice that >2000bp contigs cut; step size = 100bp had highest N50 and sum

ID1 (average assembly)[edit]

n       n:100   n:N50  min    median mean   N50    max    sum
2035    2035    122    100    140    476    1695   17800  968883  contigs_2000_100.fa
342     342     31     100    368    1337   3721   19794  457301  SAG_A_ID1_and_recruted_Meta_all_Assembly 
4428    4428    232    100    188    829    3185   19794  3674310 ../SAG_A_ID1_and_recruted_Meta_all
1742    1742    362    100    123    199    216    3710   348324  s6-8_A_ID1.contig.100up

Conclusions[edit]

  • Based on ID9 test, >2000bp contigs cut and step size 100bp had best results when using sliding cut method
  • When comparing sliding cut method and just assembly of recruited sequences and SAG contigs, sliding cut shows ~2-6x increase in sum, and approximately same (sometimes slightly lower) N50 and max
    • Sliding cut method improves recruitment assembly when compared with assembly w/o sliding cut
  • Based on both tests, when using sliding cut, recruitment showed a marked increase in assembly (i.e. higher N50, max, and sum) than original SAG contigs -> shows that recruitment helps with assembly (need to validate if assembly is accurate)
    • Recruitment w/ sliding cut shows improvement compared to SAG
  • However, when comparing sliding cut assembly and just statistics of SAG and recruited contigs (without assembly) = sliding cut shows lower sum/N50/max
    • Not expected to beat out combination without assembly because there is redundancy in sequencies

Follow-Up[edit]

  • Sam brought out the question: would the number of copies change the sum?
    • Here, test whether the number of copies changes any of the contig statistics
  • Procedure: Above, the script had 3 copies of each contig (both split and un-split)
    • 1. Change script so have 1 copy
    • 2. Change script so have 5 copies
    • 3. Run both changed scripts on ID9 with >2000bp contigs cut and step size 100bp
  • Results
n       n:100   n:N50  min    median mean   N50    max    sum
1280    1280    138    100    341    1334   3500   20981  1707966 contigs_2000_100_1_copy.fa
7819    7819    710    100    197    473    1057   21081  3703868 contigs_2000_100_3_copy.fa
7820    7820    710    100    197    473    1057   21081  3704053 contigs_2000_100_5_copy.fa
  • Conclusion
    • Increasing the number of copies above 3 copies only slightly changes the sum -> the sum plateaus with change of number of copies (but max and N50 are max at number of copies = 3 already)