Main statistics¶

  value
metric  
MIXED_read_mean_coverage 14.09
PCT_MIXED_both_tags 36.97
PCT_MIXED_both_tags_where_endreached 40.66
PCT_MINUS_both_tags_where_endreached 12.02
PCT_PLUS_both_tags_where_endreached 17.74
PCT_UNDETERMINED_either_tag 18.79
PCT_DISCORDANT 10.79
PCT_read_end_unreached 9.08
Mean_cvg 38.10
Indel_Rate 0.37
Mean_Read_Length 186.75
PF_Barcode_reads 727177953.00
PCT_PF_Reads_aligned 99.83
PCT_Chimeras 0.20
PCT_duplicates 18.19
PCT_Failed_QC_reads 0.00
PCT_failed_adapter_dimers 0.25
PCT_failed_unrecognized_start_stem 0.18
PCT_failed_unrecognized_start_loop 0.09




  • MIXED_read_mean_coverage is the coverage of reads where the tag in both the start and the end of the read were detected as MIXED
  • pct_MIXED_both_tags is the ratio of reads where both loops were detected as MIXED out of all the reads
  • pct_MIXED_both_tags_where_endreached is the ratio of reads where both loops were detected as MIXED out of the reads where the read end was reached so that the end loop could be measured

QC plots¶

No description has been provided for this image
This barplot shows the ratio of each category type in the data according to the spec in the top of the file.
The categories are reported separately for the start- and end-loops.
The end loop breakdown is shown only for the reads that reached the end loop.





No description has been provided for this image
These plots show the concordance between the strand ratio categories of the start-loop and end-loop. Each loop is assigned a category separately, and the concordance is plotted. The top plot includes all the reads, including those with END_UNREACHED, while the bottom includes reads where the end was reached only.





No description has been provided for this image
This plot shows the homopolymers called in the A, T, G and C hmers in the start loop (left) and in the T, G, C, A hmers in the end loop (right). The loops are expected to yield:
- A signal of [1 1 1 1], AGCT and GCAT for the start and end loops, for MIXED reads
- A signal of [0 2 0 2], TTCC and CCTT for the start and end loops, for MINUS-only reads
- A signal of [2 0 2 0], AAGG and GGAA for the start and end loops, for PLUS-only reads





No description has been provided for this image

About ppmSeq¶

Identifying single nucleotide variants (SNVs) is fundamental to genomics. While consensus mutation calling, requiring multiple variant-containing reads to call genetic variation, is often used, it is unsuitable in calling rare SNVs, such as in circulating tumor DNA or somatic mosaicism, where often only a single supporting read is available. Paired Plus and Minus strand Sequencing (ppmSeq), a PCR-free library preparation technology that uniquely leverages the Ultima Genomics clonal amplification process, overcomes this challenge. Here, DNA denaturation is not required prior to clonal amplification so both native strands are clonally amplified on many sequencing beads, allowing for a linear increase in duplex recovery and scalable duplex coverage without requiring unique molecular identifiers or redundant sequencing.

In ppmSeq, modified Ultima Genomics adapters containing mismatched homopolymers are used to detect reads that are the result of the mixture of the two native DNA strands. While some reads are amplicons of only the Plus or Minus strands and are generally of typical UG read SNV accuracy, the so-called Mixed reads exhibit much lower error rates, well below 1E-6, facilitating the accurate detection of rare SNVs. Artifactual mutations manifesting on one strand only are common sources of error in SNV detection from NGS. While beads that are amplicons of Plus or Minus strand only are exposed to these artifacts that would appear as high-quality reads, in Mixed beads they create an inconsistent signal that translates into a low quality base or read, preventing them from being read as false positive SNVs.

This report is generated from preprocessing of the ppmSeq sequencing data, and is intended to be used as a QC report for the library prep and sequencing run. The distribution of the MINUS/PLUS ratio, assignment of reads to categories (MIXED/MINUS/PLUS/UNDETERMINED), and with the raw calls are shown.

ppmSeq adapter version¶

The ppmSeq_v1 adapter is used in this sample. It is composed of an AAGG-AAGG loop in the start and a GGAA-GGAA loop in "
"the end of the read, so that reads are expected to ideally yield in each loop:
- TTCC and CCTT for MINUS-only reads
- AAGG and GGAA for PLUS-only reads
- AGCT and GCAT for 50% MINUS - 50% PLUS reads

Up to 2 homopolymer errors are allowed, as long as the distance from the second best fit is at least 4.

Additionally, since the end loop is at the end of the reads it is not necessarily reached, in which case the loop is "
"annotated as END_UNREACHED.





Detailed statistics¶


Statistics table: keys_to_convert
0                      stats_shortlist
1                         sorter_stats
2         strand_ratio_category_counts
3           strand_ratio_category_norm
4    strand_ratio_category_concordance
5      strand_ratio_category_consensus
6                trimmer_failure_codes
dtype: object

Statistics table: sorter_stats
metric
Mean_cvg                3.810000e+01
Indel_Rate              3.700000e-01
Mean_Read_Length        1.867500e+02
PF_Barcode_reads        7.271780e+08
PCT_PF_Reads_aligned    9.983000e+01
PCT_Chimeras            2.000000e-01
PCT_duplicates          1.819000e+01
PCT_Failed_QC_reads     0.000000e+00
Name: value, dtype: float64

Statistics table: stats_shortlist
metric
MIXED_read_mean_coverage                1.408537e+01
PCT_MIXED_both_tags                     3.696947e+01
PCT_MIXED_both_tags_where_endreached    4.066336e+01
PCT_MINUS_both_tags_where_endreached    1.202371e+01
PCT_PLUS_both_tags_where_endreached     1.773872e+01
PCT_UNDETERMINED_either_tag             1.878890e+01
PCT_DISCORDANT                          1.078531e+01
PCT_read_end_unreached                  9.084078e+00
Mean_cvg                                3.810000e+01
Indel_Rate                              3.700000e-01
Mean_Read_Length                        1.867500e+02
PF_Barcode_reads                        7.271780e+08
PCT_PF_Reads_aligned                    9.983000e+01
PCT_Chimeras                            2.000000e-01
PCT_duplicates                          1.819000e+01
PCT_Failed_QC_reads                     0.000000e+00
PCT_failed_adapter_dimers               2.538253e-01
PCT_failed_unrecognized_start_stem      1.799352e-01
PCT_failed_unrecognized_start_loop      9.323381e-02
Name: value, dtype: float64

Statistics table: strand_ratio_category_concordance
strand_ratio_category_start  strand_ratio_category_end
MIXED                        MIXED                        0.369695
                             MINUS                        0.015190
                             PLUS                         0.057858
                             END_UNREACHED                0.051359
                             UNDETERMINED                 0.095996
MINUS                        MIXED                        0.004068
                             MINUS                        0.109315
                             PLUS                         0.010558
                             END_UNREACHED                0.015908
                             UNDETERMINED                 0.026331
PLUS                         MIXED                        0.006498
                             MINUS                        0.003883
                             PLUS                         0.161273
                             END_UNREACHED                0.018783
                             UNDETERMINED                 0.019044
UNDETERMINED                 MIXED                        0.009739
                             MINUS                        0.001027
                             PLUS                         0.007375
                             END_UNREACHED                0.004790
                             UNDETERMINED                 0.011309
Name: count_norm, dtype: float64

Statistics table: strand_ratio_category_consensus
strand_ratio_category_consensus
MIXED           0.406634
MINUS           0.120237
PLUS            0.177387
UNDETERMINED    0.187889
DISCORDANT      0.107853
Name: count_norm, dtype: float64

Statistics table: strand_ratio_category_counts
strand_ratio_category_start strand_ratio_category_end strand_ratio_category_end_no_unreached
MIXED 424970082 280865488 280865488
MINUS 119677762 93200168 93200168
PLUS 150861435 170727150 170727150
END_UNREACHED 0 65420666 0
UNDETERMINED 24659175 109954982 109954982

Statistics table: strand_ratio_category_norm
strand_ratio_category_start strand_ratio_category_end strand_ratio_category_end_no_unreached
MIXED 0.590098 0.390000 0.428967
MINUS 0.166180 0.129414 0.142345
PLUS 0.209481 0.237066 0.260753
END_UNREACHED 0.000000 0.090841 0.000000
UNDETERMINED 0.034241 0.152680 0.167935

Statistics table: trimmer_failure_codes
failed_read_count total_read_count PCT_failure
segment reason
First_C no match 34 840359343 0.000004
sequence was too short 159 840359343 0.000019
Stem_start no match 1512102 840359343 0.179935
Unrecognized_End_loop sequence was too long 2059947 840359343 0.245127
Unrecognized_Start_loop sequence was too long 783499 840359343 0.093234
insert sequence was too short 2133045 840359343 0.253825
start rsq file 113181390 840359343 13.468213
sequence was too long 520713 840359343 0.061963

Statistics table: trimmer_histogram
strand_ratio_category_start loop_sequence_start strand_ratio_category_end loop_sequence_end native_adapter_length count count_norm
0 PLUS AAGGA PLUS GGAAC 1.0 96549919 0.134066
1 MINUS TTCCA MINUS CCTTC 1.0 74124550 0.102927
2 MIXED TGCA MIXED GCATTC 1.0 47382908 0.065794
3 MIXED ATGCA UNDETERMINED NaN 1.0 36098335 0.050125
4 MIXED ATGCA MIXED GCATTC 1.0 36057882 0.050069
5 MIXED ATGCA MIXED GGCATTC 1.0 34369215 0.047724
6 MIXED TGCA MIXED GGCATTC 1.0 22794375 0.031651
7 MIXED TGCA UNDETERMINED NaN 1.0 22414943 0.031125
8 MIXED ATGCA END_UNREACHED NaN NaN 17981147 0.024968
9 MINUS TTCCA UNDETERMINED NaN 1.0 16487670 0.022894
10 PLUS AAGGA PLUS GGAC 1.0 16232998 0.022541
11 MIXED ATGCA PLUS GGAAC 1.0 16200336 0.022495
12 MIXED TGCA PLUS GGAAC 1.0 15317574 0.021269
13 PLUS AAGGA UNDETERMINED NaN 1.0 13466567 0.018699
14 MIXED ATGCA MIXED GGCTC 1.0 13193497 0.018320
15 PLUS AAGGA END_UNREACHED NaN NaN 12795650 0.017768
16 MIXED TGCA MIXED GCTC 1.0 12705557 0.017642
17 MIXED ATGCA MIXED GCTC 1.0 12350077 0.017149
18 MIXED TGCA END_UNREACHED NaN NaN 12123586 0.016834
19 MINUS TTCCA END_UNREACHED NaN NaN 10398363 0.014439
20 MIXED ATGCA MIXED GTC 1.0 10352171 0.014375
21 MIXED TGCA MIXED GGCTC 1.0 9099214 0.012635
22 MIXED ATGCA MIXED GGCATC 1.0 8400699 0.011665
23 UNDETERMINED NaN UNDETERMINED NaN 1.0 8144618 0.011309
24 MIXED ATTGCA MIXED GCATTC 1.0 7534241 0.010462
25 MIXED TGCA MIXED GTC 1.0 6956489 0.009660
26 MIXED ATTGCA UNDETERMINED NaN 1.0 6228763 0.008649
27 MINUS TTCCA PLUS GGAAC 1.0 5983183 0.008308
28 MIXED ATGCA MIXED GCATC 1.0 5728368 0.007954
29 MIXED ATGCA MINUS CCTTC 1.0 4792747 0.006655
30 UNDETERMINED NaN MIXED GCATTC 1.0 4696143 0.006521
31 MIXED TGCA MINUS CCTTC 1.0 4686088 0.006507
32 MIXED ATTGCA END_UNREACHED NaN NaN 4329181 0.006011
33 MIXED ATTGCA MIXED GGCATTC 1.0 4076482 0.005660
34 MIXED ATGCA MIXED GATTC 1.0 3754425 0.005213
35 MIXED TGCA MIXED GATTC 1.0 3719897 0.005165
36 UNDETERMINED NaN END_UNREACHED NaN NaN 3377995 0.004691
37 MIXED TGCA MIXED GCATC 1.0 3281039 0.004556
38 UNDETERMINED NaN PLUS GGAAC 1.0 2816179 0.003910
39 MIXED ATGCA PLUS GGAC 1.0 2765177 0.003840
40 PLUS AAGGA MINUS CCTTC 1.0 2648343 0.003677
41 MIXED ATGCA MIXED GATC 1.0 2522530 0.003503
42 MIXED AATGCA UNDETERMINED NaN 1.0 2519505 0.003498
43 MIXED TGCA PLUS GGAC 1.0 2412285 0.003350
44 MIXED TGCA MIXED GGCATC 1.0 2284696 0.003172
45 UNDETERMINED NaN PLUS GGAC 1.0 2234964 0.003103
46 MIXED ATTGCA MIXED GCTC 1.0 1984910 0.002756
47 MIXED ATTGCA PLUS GGAAC 1.0 1869526 0.002596
48 PLUS AAGGA MIXED GCATTC 1.0 1691922 0.002349
49 MIXED ATGCA MIXED TGCTC 1.0 1489937 0.002069