Cell Free DNA WGBS Analysis Report
2021-01-14

1. Fastq Quality Control

The followings are quality control files generated by FastQC. For more detailed information, please click the hyperlinks below.

Sample: case1

case1_1_fastqc.html
case1_2_fastqc.html

Sample: case2

case2_1_fastqc.html
case2_2_fastqc.html

Sample: case3

case3_1_fastqc.html
case3_2_fastqc.html

Sample: case4

case4_1_fastqc.html
case4_2_fastqc.html


2. Adapter Detection

Sample: case1

  --adapter1:  AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG

               ||||||||||||||||||||||||||||||||||******||||||||||||||||||||||||

   Consensus:  AGATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTGAAAAAACCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC

     Quality:  346665757656656574566356536..-.0,21,++,+-+-(,,*-)+,,)(,((*)))'''((**)(#$$$$$$$##$#$#####$$%#%$$$$$##$#$#$##$$%%%$$$$$$$#$$########$######"$

Top 5 most common 9-bp 5'-kmers:
AGATCGGAA95.96%(210489)
CGATCGGAA0.41%(904)
ATATCGGAA0.23%(502)
AGATCTGAA0.20%(448)
AGATCGGGA0.18%(396) 

  --adapter2:  AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT

               ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

   Consensus:  AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATTAAAAAAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG

     Quality:  47445763340301./0-/-.44/01..,/,---.,-.,-4/+4.*,..-.)++-+,+,--,-+$$$%%%%%%%%%%%%&&&&''&&'&&&&%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%$$%$$$#$$$$$$

Top 5 most common 9-bp 5'-kmers:
AGATCGGAA95.33%(209099)
AGATCGGGA0.21%(468)
AGATCGAAG0.20%(449)
AGATCGGAG0.20%(439)
AGATCGGCA0.18%(405)

Sample: case2

  --adapter1:  AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG

               ||||||||||||||||||||||||||||||||||******||||||||||||||||||||||||

   Consensus:  AGATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACCAATCTCGTATGCCGTCTTCTGCTTGAAAAAACCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC

     Quality:  446676757656656674464354426--,-0+0,++0.,,*,&++),(*++('+'(*(((&&&'())('#$$%$$$$##$#$#####$#%#%#$$$$##$#$#$##$$%$%$%$$$#$#$#####$#$#$##"##$#"

Top 5 most common 9-bp 5'-kmers:
AGATCGGAA96.29%(200938)
CGATCGGAA0.35%(732)
ATATCGGAA0.21%(446)
AGATCTGAA0.18%(383)
AGATCGTAA0.15%(307) 

  --adapter2:  AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT

               ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

   Consensus:  AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATTAAAAAAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG

     Quality:  3733475224/201-//-/--43..0-.,.+-,,-,--,-3/+3.*+.-,.)**,+,++,,+,*$$$%%%%%%%%%%%%&&&&'&&&'&&&&%%%%%&%%%%%%%%%%%%%%%%%%%%%%%%%%%%$$$$$$$$$%$$%

Top 5 most common 9-bp 5'-kmers:
AGATCGGAA94.64%(197494)
AGATCGGGA0.24%(507)
AGATCGGAG0.23%(471)
AGATCGAAG0.22%(462)
AGATCGGCA0.21%(443)

Sample: case3

  --adapter1:  AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG

               ||||||||||||||||||||||||||||||||||******||||||||||||||||||||||||

   Consensus:  AGATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGGCATCTCGTATGCCGTCTTCTGCTTGAAAAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGT

     Quality:  6066.1377152252411001133000010//1/.00./.0.-',.--.,+,-,+++)*,*)*+)+((%'**+++++++++++++++++++++++++++++++++,+++,,,,,,,,,%$$########"#""""""##

Top 5 most common 9-bp 5'-kmers:
AGATCGGAA90.28%(338989)
AGATTGGAA2.38%(8921)
ATATCGGAA1.55%(5831)
AGATCTGAA1.12%(4214)
AGATAGGAA0.98%(3694) 

  --adapter2:  AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT

               ||||||||||||||||||||||||||||||||||| | | || ||  |  |      |

   Consensus:  AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGGTGTGGGGGGGGGGGGGGGGGGTGAAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGAAAAAAAAAAAAAA

     Quality:  6/541/15312202011.1-//00+,*.&/(/%#+#&%$$..$..%&.&$-&%#%$$$#####%'''(((((()))*****)*)****))))))***************+++++++++$$##""""""""##""""""#

Top 5 most common 9-bp 5'-kmers:
AGATCGGAA89.97%(337863)
ATATCGGAA1.52%(5699)
AGATCAGAA1.27%(4766)
AGATCTGAA0.96%(3591)
AGATAGGAA0.81%(3038)

Sample: case4

  --adapter1:  AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG

               ||||||||||||||||||||||||||||||||||******||||||||||||||||||||||||

   Consensus:  AGATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACCAATCTCGTATGCCGTCTTCTGCTTGAAAAAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGTGAATTTTTTTTTTT

     Quality:  6/66.23770513524211110330/111///0/./0/001--',.'&,$%+*&&%*#%*%$(($)*)#$(*+,,------,,,,,,,,,,,,,,,,+,,,,,,,,,,,,,,,,,,,,%$###"""""""#"#######

Top 5 most common 9-bp 5'-kmers:
AGATCGGAA90.70%(367234)
ATATCGGAA2.09%(8448)
AGATTGGAA2.03%(8229)
AGATCTGAA0.83%(3373)
AGATAGGAA0.63%(2533) 

  --adapter2:  AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT

               ||||||||||||||||||||||||||||||||| | |   || ||  |  |      |

   Consensus:  AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTTGGTGGGGGGGGGGGGGGGGGGGGTGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGNAAAAAAAAAAAAAAAAT

     Quality:  7/642137523323122.2-0022+-+/&0'0$#+#%&#%/0%/0%'/&%/'&$&%$$$###$%'(((((())*+++++++*++++++**********++++++++++++++++++++$##""#"##"########$##

Top 5 most common 9-bp 5'-kmers:
AGATCGGAA92.63%(374997)
ATATCGGAA1.66%(6714)
AGATCAGAA1.00%(4044)
AGATAGGAA0.51%(2052)
AGATCTGAA0.39%(1576)


3. Bismark Alignment

Sample: case1

Final Alignment Report
  • Sequence pairs analysed in total: 4988795
  • Number of paired-end alignments with a unique best hit: 2654765
  • Mapping efficiency: 53.2%
  • Sequence pairs with no alignments under any condition: 2237074
  • Sequence pairs did not map uniquely: 96956
  • Sequence pairs which were discarded because genomic sequence could not be extracted: 0
  • Number of sequence pairs with unique best (first) alignment came from the bowtie output:
    CT/GA/CT:1327882((converted) top strand)
    GA/CT/CT:0(complementary to (converted) top strand)
    GA/CT/GA:0(complementary to (converted) bottom strand)
    CT/GA/GA:1326883((converted) bottom strand)
  • Number of alignments to (merely theoretical) complementary strands being rejected in total: 0
Final Cytosine Methylation Report
  • Total number of C's analysed: 152602870
  • Total methylated C's in CpG context: 5321803
  • Total methylated C's in CHG context: 834006
  • Total methylated C's in CHH context: 3805489
  • Total methylated C's in Unknown context: 130443
  • Total unmethylated C's in CpG context: 1184136
  • Total unmethylated C's in CHG context: 31189479
  • Total unmethylated C's in CHH context: 110267957
  • Total unmethylated C's in Unknown context: 230412
  • C methylated in CpG context: 81.8%
  • C methylated in CHG context: 2.6%
  • C methylated in CHH context: 3.3%
  • Estimated Bisulfite Conversion Rate: 96.7%

Sample: case2

Final Alignment Report
  • Sequence pairs analysed in total: 4988485
  • Number of paired-end alignments with a unique best hit: 2585946
  • Mapping efficiency: 51.8%
  • Sequence pairs with no alignments under any condition: 2310412
  • Sequence pairs did not map uniquely: 92127
  • Sequence pairs which were discarded because genomic sequence could not be extracted: 0
  • Number of sequence pairs with unique best (first) alignment came from the bowtie output:
    CT/GA/CT:1293233((converted) top strand)
    GA/CT/CT:0(complementary to (converted) top strand)
    GA/CT/GA:0(complementary to (converted) bottom strand)
    CT/GA/GA:1292713((converted) bottom strand)
  • Number of alignments to (merely theoretical) complementary strands being rejected in total: 0
Final Cytosine Methylation Report
  • Total number of C's analysed: 144496702
  • Total methylated C's in CpG context: 4989749
  • Total methylated C's in CHG context: 807080
  • Total methylated C's in CHH context: 3714455
  • Total methylated C's in Unknown context: 126808
  • Total unmethylated C's in CpG context: 1052508
  • Total unmethylated C's in CHG context: 29386879
  • Total unmethylated C's in CHH context: 104546031
  • Total unmethylated C's in Unknown context: 222035
  • C methylated in CpG context: 82.6%
  • C methylated in CHG context: 2.7%
  • C methylated in CHH context: 3.4%
  • Estimated Bisulfite Conversion Rate: 96.7%

Sample: case3

Final Alignment Report
  • Sequence pairs analysed in total: 4983058
  • Number of paired-end alignments with a unique best hit: 3558097
  • Mapping efficiency: 71.4%
  • Sequence pairs with no alignments under any condition: 1218791
  • Sequence pairs did not map uniquely: 206170
  • Sequence pairs which were discarded because genomic sequence could not be extracted: 0
  • Number of sequence pairs with unique best (first) alignment came from the bowtie output:
    CT/GA/CT:1766520((converted) top strand)
    GA/CT/CT:0(complementary to (converted) top strand)
    GA/CT/GA:0(complementary to (converted) bottom strand)
    CT/GA/GA:1791577((converted) bottom strand)
  • Number of alignments to (merely theoretical) complementary strands being rejected in total: 0
Final Cytosine Methylation Report
  • Total number of C's analysed: 161117086
  • Total methylated C's in CpG context: 5868066
  • Total methylated C's in CHG context: 990549
  • Total methylated C's in CHH context: 2656923
  • Total methylated C's in Unknown context: 899
  • Total unmethylated C's in CpG context: 1335565
  • Total unmethylated C's in CHG context: 32255260
  • Total unmethylated C's in CHH context: 118010723
  • Total unmethylated C's in Unknown context: 46478
  • C methylated in CpG context: 81.5%
  • C methylated in CHG context: 3.0%
  • C methylated in CHH context: 2.2%
  • Estimated Bisulfite Conversion Rate: 97.6%

Sample: case4

Final Alignment Report
  • Sequence pairs analysed in total: 4993750
  • Number of paired-end alignments with a unique best hit: 3678864
  • Mapping efficiency: 73.7%
  • Sequence pairs with no alignments under any condition: 1111269
  • Sequence pairs did not map uniquely: 203617
  • Sequence pairs which were discarded because genomic sequence could not be extracted: 1
  • Number of sequence pairs with unique best (first) alignment came from the bowtie output:
    CT/GA/CT:1830602((converted) top strand)
    GA/CT/CT:0(complementary to (converted) top strand)
    GA/CT/GA:0(complementary to (converted) bottom strand)
    CT/GA/GA:1848261((converted) bottom strand)
  • Number of alignments to (merely theoretical) complementary strands being rejected in total: 0
Final Cytosine Methylation Report
  • Total number of C's analysed: 169749025
  • Total methylated C's in CpG context: 5816254
  • Total methylated C's in CHG context: 1289866
  • Total methylated C's in CHH context: 4734423
  • Total methylated C's in Unknown context: 1125
  • Total unmethylated C's in CpG context: 1463517
  • Total unmethylated C's in CHG context: 33872763
  • Total unmethylated C's in CHH context: 122572202
  • Total unmethylated C's in Unknown context: 47956
  • C methylated in CpG context: 79.9%
  • C methylated in CHG context: 3.7%
  • C methylated in CHH context: 3.7%
  • Estimated Bisulfite Conversion Rate: 96.3%

4. Qualimap

The followings are Qualimap reports. For more detailed information, please click the hyperlinks below.

Sample: case1.pair1

qualimapReport.html

Sample: case2.pair1

qualimapReport.html

Sample: case3.pair1

qualimapReport.html

Sample: case4.pair1

qualimapReport.html

5. Alignment Duplication Analysis

Sample: case1

  • Total number duplicated alignments removed: 35919 (1.35%)
  • Total count of deduplicated leftover sequences: 2618846 (98.65% of total)

Sample: case2

  • Total number duplicated alignments removed: 33321 (1.29%)
  • Total count of deduplicated leftover sequences: 2552625 (98.71% of total)

Sample: case3

  • Total number duplicated alignments removed: 74114 (2.08%)
  • Total count of deduplicated leftover sequences: 3483983 (97.92% of total)

Sample: case4

  • Total number duplicated alignments removed: 88708 (2.41%)
  • Total count of deduplicated leftover sequences: 3590155 (97.59% of total)

6. Fragment Length Distribution

Fragment length distribution of the samples:
/opt/tsinghua/zhangwei/Pipeline_test/o_WGBS-PE/report_result/Fragment_Length/length_distribution.png

7. Copy Number Variation Analysis

The followings are plots generated by cnvPlot. For more detailed information, please click the hyperlinks below.

Sample: case1.pair1

case1.pair1_diagram.pdf
case1.pair1_diagram.pdf

Sample: case2.pair1

case2.pair1_diagram.pdf
case2.pair1_diagram.pdf

Sample: case3.pair1

case3.pair1_diagram.pdf
case3.pair1_diagram.pdf

Sample: case4.pair1

case4.pair1_diagram.pdf
case4.pair1_diagram.pdf


8. CNV Heatmap

For more detailed information, please click the hyperlinks below.
heatmap.pdf