单细胞数据 mkfastq | 10x Genomics

 

除了刚接触10x的那会儿，还真没怎么亲自倒腾过fastq的制作。

正常从测序商那里拿到的应该是bcl的原始数据，需要自己做一步bcl2fastq。

后面大家都觉得这一步太麻烦了，没必要，所以大部分测序商也就自己做了。

这也是对的，生信分析的起点是fastq，不应该是bcl，数据保存的最原始格式也应该是压缩了的fastq，而不是过于原始的bcl。

 

现在有批data，fastq被搞不见了，只有原始的bcl还保存着，于是就要回来了自己转fastq。

10x的每一个工具都有自己的mkfastq的教程

 

运行代码后发现我的BCL文件居然也不是完整的。

一个标准的bcl目录必须包含以下文件：

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cellranger-tiny-bcl-1.2.0 ├── Config │   ├── h35kcbcxy_Oct-20-16\ 15-51-48_Effective.cfg │   ├── HiSeqControlSoftware.Options.cfg │   ├── RTAStart.bat │   └── Variability_HiSeq_C.bin ├── Data │   └── Intensities ├── InterOp │   ├── ControlMetricsOut.bin │   ├── CorrectedIntMetricsOut.bin │   ├── ErrorMetricsOut.bin │   ├── ExtractionMetricsOut.bin │   ├── ImageMetricsOut.bin │   ├── QMetricsOut.bin │   └── TileMetricsOut.bin ├── RTAComplete.txt ├── RunInfo.xml └── runParameters.xml

　

运行需要一个samplesheet文件

When you plan an experiment, you should know the name of the sample index set used for each sample, which comes from the reagent kit (such as "SI-TT-A2"). For each sample, enter its lane, sample name, and sample index set into the Illumina bcl2fastq sample sheet. Here is a bcl2fastq sample sheet for a HiSeq 2500:

 

For example, a Multiome GEX library prepared with the Dual Index Kit TT Set A, well A1 can be specified in the sample sheet as "SI-TT-A1", and cellranger-arc mkfastq will recognize the i7 and i5 indices as GTAACATGCG and AGTGTTACCT, respectively.

Similarly for a Multiome ATAC library prepared with Single Index Kit N Set A, well A1 can be specified in the sample sheet as "SI-NA-A1", and cellranger-arc mkfastq will recognize the four i7 indexes (AAACGGCG, CCTACCAT, GGCGTTTC, and TTGTAAGA) and merge the resulting FASTQ files.　

 

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1	conda install -c dranew bcl2fastq

　　

 

参考：

• Generating FASTQs with cellranger-arc mkfastq
• Running cellranger mkfastq