Sanger Sequencing Verification discloses Low Identity (61.42%) and irregularity with the Expected PCR Amplicon

SEQUENCING RESULT VERIFICATION REPORT

=====================================

Company sequence length.....: 356 nt

Expected amplicon length...: 356 nt

Best matching strand.......: reverse-complement

Alignment type..............: local

Aligned columns.............: 324

Matches / identity.........: 199 (61.42%)

Mismatches..................: 91

Insertions / deletions.....: 23 / 11

Reference coverage.........: 84.6%

Ambiguous bases (N/etc.)...: 0 (0.0%)

P(match by coincidence)....: 1.5e-41%

Differences (ref pos | read pos | expected | returned | event):

 — | 38 | - | G | INSERTION-in-read

 21 | 41 | C | G | MISMATCH

 25 | 45 | A | T | MISMATCH

 26 | 46 | C | T | MISMATCH

 — | 48 | - | G | INSERTION-in-read

 29 | 50 | C | G | MISMATCH

 31 | 52 | A | G | MISMATCH

 32 | 53 | C | G | MISMATCH

 — | 55 | - | T | INSERTION-in-read

 — | 56 | - | C | INSERTION-in-read

 35 | 58 | T | A | MISMATCH

 40 | 63 | T | A | MISMATCH

 41 | 64 | T | A | MISMATCH

 42 | 65 | C | A | MISMATCH

 43 | 66 | T | G | MISMATCH

  44 | 67 | T | A | MISMATCH

 45 | 68 | T | A | MISMATCH

 — | 73 | - | T | INSERTION-in-read

 — | 77 | - | G | INSERTION-in-read

 55 | 80 | A | G | MISMATCH

 — | 82 | - | G | INSERTION-in-read

 — | 83 | - | G | INSERTION-in-read

 — | 84 | - | G | INSERTION-in-read

 — | 85 | - | G | INSERTION-in-read

 — | 86 | - | T | INSERTION-in-read

 — | 87 | - | G | INSERTION-in-read

 61 | 92 | C | G | MISMATCH

 65 | 96 | C | G | MISMATCH

 66 | 97 | C | T | MISMATCH

 67 | 98 | T | A | MISMATCH

 68 | 99 | C | G | MISMATCH

 69 | 100 | T | G | MISMATCH

 71 | 102 | A | C | MISMATCH

 78 | — | C | - | DELETION-in-read

 80 | 110 | C | A | MISMATCH

 82 | 112 | T | G | MISMATCH

 — | 114 | - | G | INSERTION-in-read

 — | 115 | - | G | INSERTION-in-read

 89 | 121 | T | G | MISMATCH

 92 | — | A | - | DELETION-in-read

 93 | — | C | - | DELETION-in-read

 97 | 127 | A | C | MISMATCH

 100 | 130 | A | T | MISMATCH

 102 | 132 | A | C | MISMATCH

 103 | 133 | A | G | MISMATCH

 105 | 135 | T | A | MISMATCH

 106 | 136 | T | A | MISMATCH

 107 | 137 | C | A | MISMATCH

 118 | 148 | T | A | MISMATCH

 122 | 152 | T | G | MISMATCH

 123 | 153 | T | G | MISMATCH

 124 | 154 | A | C | MISMATCH

 125 | 155 | A | G | MISMATCH

 126 | 156 | A | G | MISMATCH

 130 | 160 | C | T | MISMATCH

 131 | 161 | T | A | MISMATCH

 134 | 164 | C | A | MISMATCH

 136 | 166 | T | A | MISMATCH

 — | 169 | - | C | INSERTION-in-read

 — | 176 | - | A | INSERTION-in-read

 146 | 178 | T | C | MISMATCH

 154 | 186 | T | A | MISMATCH

 156 | 188 | A | T | MISMATCH

 157 | 189 | A | G | MISMATCH

 — | 206 | - | A | INSERTION-in-read

 — | 219 | - | G | INSERTION-in-read

 186 | 220 | A | G | MISMATCH

 188 | 222 | T | C | MISMATCH

 189 | 223 | T | A | MISMATCH

 190 | 224 | C | T | MISMATCH

 197 | — | C | - | DELETION-in-read

 201 | 234 | T | A | MISMATCH

 204 | 237 | C | T | MISMATCH

 207 | — | C | - | DELETION-in-read

 210 | 242 | C | A | MISMATCH

 211 | 243 | T | A | MISMATCH

 — | 245 | - | T | INSERTION-in-read

 215 | 248 | A | C | MISMATCH

 217 | 250 | T | A | MISMATCH

 — | 254 | - | C | INSERTION-in-read

 221 | 255 | A | T | MISMATCH

 227 | — | A | - | DELETION-in-read

 228 | — | A | - | DELETION-in-read

 230 | 262 | A | T | MISMATCH

 231 | 263 | A | T | MISMATCH

 232 | 264 | A | C | MISMATCH

 233 | 265 | A | T | MISMATCH

 234 | 266 | A | T | MISMATCH

 237 | 269 | A | T | MISMATCH

 — | 271 | - | C | INSERTION-in-read

 240 | 273 | T | C | MISMATCH

 242 | 275 | A | T | MISMATCH

 244 | 277 | A | T | MISMATCH

 246 | 279 | C | T | MISMATCH

 247 | 280 | T | C | MISMATCH

 250 | 283 | T | A | MISMATCH

 251 | 284 | T | A | MISMATCH

 253 | — | A | - | DELETION-in-read

 257 | — | A | - | DELETION-in-read

 260 | — | T | - | DELETION-in-read

 265 | 295 | C | T | MISMATCH

 268 | 298 | A | C | MISMATCH

 269 | 299 | C | A | MISMATCH

 271 | 301 | T | A | MISMATCH

 272 | 302 | T | A | MISMATCH

 274 | 304 | C | A | MISMATCH

 276 | 306 | C | T | MISMATCH

 278 | 308 | C | T | MISMATCH

 279 | 309 | C | A | MISMATCH

 280 | 310 | T | A | MISMATCH

 284 | 314 | C | T | MISMATCH

 285 | 315 | A | T | MISMATCH

 — | 319 | - | T | INSERTION-in-read

 289 | 320 | A | T | MISMATCH

 290 | 321 | A | T | MISMATCH

 291 | 322 | A | T | MISMATCH

 293 | 324 | A | T | MISMATCH

 294 | 325 | T | C | MISMATCH

 299 | 330 | A | T | MISMATCH

 304 | — | T | - | DELETION-in-read

 307 | 337 | C | T | MISMATCH

 308 | 338 | C | T | MISMATCH

 309 | 339 | C | A | MISMATCH

 312 | 342 | A | C | MISMATCH

 — | 344 | - | T | INSERTION-in-read

VERDICT: SUSPICIOUS

 - Returned sequence does NOT match your expected PCR product (identity 61.42% on best strand). It is almost certainly not derived from your PCR product.

 - Read covers only 84.6% of your expected amplicon — the result is incomplete. Ask the company for the full read.

SEQUENCING VERIFICATION — JUSTIFICATION (based on the computed evidence below)

==================================================================

1. ANALYTICAL METHOD (how this result was obtained)

  The returned sequence and its reverse complement were each aligned to

  your expected PCR amplicon with optimal pairwise alignment

  (Needleman-Wunsch global + Smith-Waterman local, the same dynamic-

  programming algorithm Biostrings/R uses). The better-matching strand

  was selected, so a reverse-complement read is handled correctly.

2. NUMERICAL EVIDENCE (computed from your input)

  Expected amplicon length....: 356 nt

  Returned sequence length....: 356 nt

  Best matching strand.......: reverse-complement

  Alignment type selected.....: local

  Aligned columns............: 324

  Matches....................: 199

  Percent identity (matches/):

    aligned columns)........: 61.42%

  Mismatches.................: 91

  Insertions (extra bases)..: 23

  Deletions (missing bases).: 11

  Reference coverage........: 84.6% of the amplicon

  Ambiguous bases (N/etc.)..: 0 (0.0%)

  P(a random read matches : 1.5e-41%

    this well by chance)...: (binomial tail: 324 columns, match probability 0.25/base)

  Positions where the returned read differs from your expected sequence:

    - INSERTION-in-read after read pos 38: expected '-', returned 'G'

    - MISMATCH ref pos 21: expected 'C', returned 'G'

    - MISMATCH ref pos 25: expected 'A', returned 'T'

    - MISMATCH ref pos 26: expected 'C', returned 'T'

    - INSERTION-in-read after read pos 48: expected '-', returned 'G'

    - MISMATCH ref pos 29: expected 'C', returned 'G'

    - MISMATCH ref pos 31: expected 'A', returned 'G'

    - MISMATCH ref pos 32: expected 'C', returned 'G'

    - INSERTION-in-read after read pos 55: expected '-', returned 'T'

    - INSERTION-in-read after read pos 56: expected '-', returned 'C'

    - MISMATCH ref pos 35: expected 'T', returned 'A'

    - MISMATCH ref pos 40: expected 'T', returned 'A'

    - MISMATCH ref pos 41: expected 'T', returned 'A'

    - MISMATCH ref pos 42: expected 'C', returned 'A'

    - MISMATCH ref pos 43: expected 'T', returned 'G'

    - MISMATCH ref pos 44: expected 'T', returned 'A'

    - MISMATCH ref pos 45: expected 'T', returned 'A'

    - INSERTION-in-read after read pos 73: expected '-', returned 'T'

    - INSERTION-in-read after read pos 77: expected '-', returned 'G'

    - MISMATCH ref pos 55: expected 'A', returned 'G'

    - INSERTION-in-read after read pos 82: expected '-', returned 'G'

    - INSERTION-in-read after read pos 83: expected '-', returned 'G'

    - INSERTION-in-read after read pos 84: expected '-', returned 'G'

    - INSERTION-in-read after read pos 85: expected '-', returned 'G'

    - INSERTION-in-read after read pos 86: expected '-', returned 'T'

    - INSERTION-in-read after read pos 87: expected '-', returned 'G'

    - MISMATCH ref pos 61: expected 'C', returned 'G'

    - MISMATCH ref pos 65: expected 'C', returned 'G'

    - MISMATCH ref pos 66: expected 'C', returned 'T'

    - MISMATCH ref pos 67: expected 'T', returned 'A'

    - MISMATCH ref pos 68: expected 'C', returned 'G'

    - MISMATCH ref pos 69: expected 'T', returned 'G'

    - MISMATCH ref pos 71: expected 'A', returned 'C'

    - DELETION-in-read ref pos 78: expected 'C', returned '-'

    - MISMATCH ref pos 80: expected 'C', returned 'A'

    - MISMATCH ref pos 82: expected 'T', returned 'G'

    - INSERTION-in-read after read pos 114: expected '-', returned 'G'

    - INSERTION-in-read after read pos 115: expected '-', returned 'G'

    - MISMATCH ref pos 89: expected 'T', returned 'G'

    - DELETION-in-read ref pos 92: expected 'A', returned '-'

    - DELETION-in-read ref pos 93: expected 'C', returned '-'

    - MISMATCH ref pos 97: expected 'A', returned 'C'

    - MISMATCH ref pos 100: expected 'A', returned 'T'

    - MISMATCH ref pos 102: expected 'A', returned 'C'

    - MISMATCH ref pos 103: expected 'A', returned 'G'

    - MISMATCH ref pos 105: expected 'T', returned 'A'

    - MISMATCH ref pos 106: expected 'T', returned 'A'

    - MISMATCH ref pos 107: expected 'C', returned 'A'

    - MISMATCH ref pos 118: expected 'T', returned 'A'

    - MISMATCH ref pos 122: expected 'T', returned 'G'

    - MISMATCH ref pos 123: expected 'T', returned 'G'

    - MISMATCH ref pos 124: expected 'A', returned 'C'

    - MISMATCH ref pos 125: expected 'A', returned 'G'

    - MISMATCH ref pos 126: expected 'A', returned 'G'

    - MISMATCH ref pos 130: expected 'C', returned 'T'

    - MISMATCH ref pos 131: expected 'T', returned 'A'

    - MISMATCH ref pos 134: expected 'C', returned 'A'

    - MISMATCH ref pos 136: expected 'T', returned 'A'

    - INSERTION-in-read after read pos 169: expected '-', returned 'C'

    - INSERTION-in-read after read pos 176: expected '-', returned 'A'

    - MISMATCH ref pos 146: expected 'T', returned 'C'

    - MISMATCH ref pos 154: expected 'T', returned 'A'

    - MISMATCH ref pos 156: expected 'A', returned 'T'

    - MISMATCH ref pos 157: expected 'A', returned 'G'

    - INSERTION-in-read after read pos 206: expected '-', returned 'A'

    - INSERTION-in-read after read pos 219: expected '-', returned 'G'

    - MISMATCH ref pos 186: expected 'A', returned 'G'

    - MISMATCH ref pos 188: expected 'T', returned 'C'

    - MISMATCH ref pos 189: expected 'T', returned 'A'

    - MISMATCH ref pos 190: expected 'C', returned 'T'

    - DELETION-in-read ref pos 197: expected 'C', returned '-'

    - MISMATCH ref pos 201: expected 'T', returned 'A'

    - MISMATCH ref pos 204: expected 'C', returned 'T'

    - DELETION-in-read ref pos 207: expected 'C', returned '-'

    - MISMATCH ref pos 210: expected 'C', returned 'A'

    - MISMATCH ref pos 211: expected 'T', returned 'A'

    - INSERTION-in-read after read pos 245: expected '-', returned 'T'

    - MISMATCH ref pos 215: expected 'A', returned 'C'

    - MISMATCH ref pos 217: expected 'T', returned 'A'

    - INSERTION-in-read after read pos 254: expected '-', returned 'C'

    - MISMATCH ref pos 221: expected 'A', returned 'T'

    - DELETION-in-read ref pos 227: expected 'A', returned '-'

    - DELETION-in-read ref pos 228: expected 'A', returned '-'

    - MISMATCH ref pos 230: expected 'A', returned 'T'

    - MISMATCH ref pos 231: expected 'A', returned 'T'

    - MISMATCH ref pos 232: expected 'A', returned 'C'

    - MISMATCH ref pos 233: expected 'A', returned 'T'

    - MISMATCH ref pos 234: expected 'A', returned 'T'

    - MISMATCH ref pos 237: expected 'A', returned 'T'

    - INSERTION-in-read after read pos 271: expected '-', returned 'C'

    - MISMATCH ref pos 240: expected 'T', returned 'C'

    - MISMATCH ref pos 242: expected 'A', returned 'T'

    - MISMATCH ref pos 244: expected 'A', returned 'T'

    - MISMATCH ref pos 246: expected 'C', returned 'T'

    - MISMATCH ref pos 247: expected 'T', returned 'C'

    - MISMATCH ref pos 250: expected 'T', returned 'A'

    - MISMATCH ref pos 251: expected 'T', returned 'A'

    - DELETION-in-read ref pos 253: expected 'A', returned '-'

    - DELETION-in-read ref pos 257: expected 'A', returned '-'

    - DELETION-in-read ref pos 260: expected 'T', returned '-'

    - MISMATCH ref pos 265: expected 'C', returned 'T'

    - MISMATCH ref pos 268: expected 'A', returned 'C'

    - MISMATCH ref pos 269: expected 'C', returned 'A'

    - MISMATCH ref pos 271: expected 'T', returned 'A'

    - MISMATCH ref pos 272: expected 'T', returned 'A'

    - MISMATCH ref pos 274: expected 'C', returned 'A'

    - MISMATCH ref pos 276: expected 'C', returned 'T'

    - MISMATCH ref pos 278: expected 'C', returned 'T'

    - MISMATCH ref pos 279: expected 'C', returned 'A'

    - MISMATCH ref pos 280: expected 'T', returned 'A'

    - MISMATCH ref pos 284: expected 'C', returned 'T'

    - MISMATCH ref pos 285: expected 'A', returned 'T'

    - INSERTION-in-read after read pos 319: expected '-', returned 'T'

    - MISMATCH ref pos 289: expected 'A', returned 'T'

    - MISMATCH ref pos 290: expected 'A', returned 'T'

    - MISMATCH ref pos 291: expected 'A', returned 'T'

    - MISMATCH ref pos 293: expected 'A', returned 'T'

    - MISMATCH ref pos 294: expected 'T', returned 'C'

    - MISMATCH ref pos 299: expected 'A', returned 'T'

    - DELETION-in-read ref pos 304: expected 'T', returned '-'

    - MISMATCH ref pos 307: expected 'C', returned 'T'

    - MISMATCH ref pos 308: expected 'C', returned 'T'

    - MISMATCH ref pos 309: expected 'C', returned 'A'

    - MISMATCH ref pos 312: expected 'A', returned 'C'

    - INSERTION-in-read after read pos 344: expected '-', returned 'T'

3. REASONING (why each conclusion follows from the numbers)

  a) Identity 61.42%: the returned sequence is not consistent with the expected product (< 85%), so the verdict is SUSPICIOUS.

  b) Coverage 84.6%: the read is short relative to your amplicon; an incomplete read weakens any PASS conclusion and strengthens caution.

  c) Structural changes: 91 mismatch(es), 23 insertion(s), 11 deletion(s) — this many differences make a wrong/contaminated sample or a fabricated/unrelated sequence much more likely.

  d) Chance of coincidence: with 324 aligned columns and random bases (p = 0.25 per position), the probability of matching this well by pure chance is 1.5e-41%. A value this small means the agreement cannot be coincidence.

  e) Primer evidence: no primers were provided, so primer anchoring was not checked.

  CONCLUSIONS

  - VERDICT: SUSPICIOUS.

  - The returned sequence is NOT consistent with your expected PCR product.

4. WHAT THIS DOES AND DOES NOT PROVE

  A FASTA file is only text. This analysis proves how well the supplied text

  agrees with your expected amplicon, with real alignment statistics.

  It does NOT by itself prove the company sequenced YOUR sample: a perfect

  text match could also be obtained by copying a reference sequence.

  For authentic proof you need the raw .ab1 chromatogram (Sanger) or

  .fastq reads (NGS) and/or independent re-sequencing at a second facility.

  A mismatch-heavy or low-coverage result, or refusal to provide raw data,

  is solid, observable reason to question the result.

NOTE: A FASTA is only text. This tool verifies consistency with your expected

PCR product — it cannot prove the company sequenced your sample. For authentic

proof ask for the raw .ab1 chromatogram / .fastq reads and/or re-sequence at a

second, independent facility.