Sanger 测序峰图查看与克隆验证
拖入 .ab1 峰图查看测序结果，再拖入质粒图谱，直接判断克隆对不对。
测序公司返回的 .ab1、.abi 或 .scf 峰图文件，在这里直接打开：四色峰图、碱基序列、Phred 质量值、可用读长和双峰诊断一次显示，可以修改碱基、裁剪两端，并导出 FASTA、FASTQ 或 SCF。打开峰图后，把构建好的质粒图谱（SnapGene .dna、GenBank 或 FASTA）拖到峰图下方，读段会与图谱比对：列出每个错配、插入和缺失在图谱上的坐标，标出读段覆盖了哪些元件，并说明每个差异对蛋白的影响——同义、错义还是提前终止。一次拖入多个峰图可以整板筛选克隆，正反向两条读段可以拼接成一条一致序列。
工具界面目前为英文：拖入文件或粘贴序列即可，结果中的数字与图表和英文版完全相同。
What do you need to verify?
All three workflows are here. Choose a task now, or carry a trace result into the next step without entering it twice.
Assembly, mixed-peak and construct checks send the derived base calls, quality scores and dye-channel arrays to the server — and, for the construct check, the template and primers you give it.
The viewer decodes the raw ABIF (.ab1 / .abi) chromatogram: the four processed dye channels are drawn as an electropherogram (G black, A green, C blue, T red) with the called bases positioned over their peaks. Read length and mean Phred quality summarize the read; export the base calls as FASTA or the trace as an SVG/PNG figure. Low peaks, broad or overlapping peaks, and stretches of low quality near the ends are the usual signs to trim before downstream analysis.
Got the reverse read too? Assemble them
Two traces that overlap in the middle are one insert sequenced from both ends. This joins them into a single consensus without needing a reference: orientation comes from the overlaps, the ends are quality-trimmed, and every position the reads disagree on is listed rather than averaged away. Assembly is the feature that separates free Chromas from paid ChromasPro, and it is on this page.
Parsed in your browser — only the base calls and quality scores are sent. Pick the forward and reverse reads together.
0 pasted reads. A pasted read has no quality scores, so its ends are not trimmed. Pasted reads and uploaded traces are assembled together.
Upload two or more traces, or paste the reads, to assemble them into one contig.
Double peaks? Read what the basecaller did not report
A basecaller reports one base per position and stops early. This reads the same trace for the second dye under a called base — a heterozygote, or a mixed colony — and for the bases still visible in the scan past the point base calling gave up. Both are ordinary reasons a read looks wrong when the DNA is fine.
Second peak / first peak. The default is deliberately conservative.
Open a trace above, and this reads what is under its base calls.
No map of the finished plasmid? Check the read against the PCR
Comparing a read needs a reference. If what you have is the template and the two primers that made the insert, this rebuilds the insert from them, finds it in the read in either orientation, and reports identity, coverage and every mismatch — and, given the frame, whether a premature stop crept in.
The insert is re-derived from these three rather than taken from you, which is the point: if the primers you recorded do not produce what you think they produce, that shows up here instead of being assumed.
Design, edit, and verify complete constructsSeqStudio combines sequence editing and annotation, plasmid maps, primer/cloning/CRISPR design, Sanger verification, and GenBank/SnapGene files in one workspace.
使用步骤
- 1把 .ab1 / .abi / .scf 文件拖到下方，或点击选择文件。
- 2查看峰图、质量和诊断结果；需要时修改或裁剪碱基。
- 3在峰图下方的 “Is the clone right?” 一栏拖入你的质粒图谱，读取比对结论和每个差异的影响。
常见问题
没有 SnapGene，怎么打开 .ab1 文件？
把文件拖到本页即可。峰图在浏览器标签页里直接打开，Windows、Mac 和 Linux 都一样，打开后还能导出 FASTA 或 FASTQ。
一条 Sanger 读段只覆盖质粒的一部分，比对结论可信吗？
图谱比读段长得多时，判定以读段本身为准：读段 90% 以上的长度落在图谱上才下结论，而且结论只针对读段覆盖的那一段。结果会写明覆盖到图谱上的哪些元件，例如“完整覆盖 AmpR”。
怎么判断一个差异是测序错误还是真实突变？
先回到峰图上看：读段两端和低质量区域的错配，常常是碱基判读错误。比对表格会写明每个差异对蛋白的影响；落在编码区、造成移码或提前终止的差异，建议用另一条读段确认后再决定是否保留克隆。
更多中文入口见 SeqBench 中文。