cellranger atac 操作笔记-3：count 输出文件解读（2-2）_scanpy fragments.tsv.gz

作者：AllinToyou | 2024-05-19 10:19:15

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scanpy fragments.tsv.gz

3. 细胞barcode质控信息，singlecell.csv

包含barcode中的片段质控信息，barcode中signal信号，与TSS重叠的片段数量等各种指标信息。

singlecell.csv文件纵坐标为barcode, total, duplicate, chimeric, unmapped, lowmapq, mitochondrial, nonprimary, passed_filters, is__cell_barcode, excluded_reason, TSS_fragments, DNase_sensitive_region_fragments, enhancer_region_fragments, promoter_region_fragments, on_target_fragments, blacklist_region_fragments, peak_region_fragments, peak_region_cutsites等18指标；横坐标为所有barcode序列，包含未质控barcode，总行数超40W。需要注意通过不同pipeline输出的结果指标数量存在一定区别。


# 文件内容
head singlecell.csv 
barcode,total,duplicate,chimeric,unmapped,lowmapq,mitochondrial,nonprimary,passed_filters,is__cell_barcode,excluded_reason,TSS_fragments,DNase_sensitive_region_fragments,enhancer_region_fragments,promoter_region_fragments,on_target_fragments,blacklist_region_fragments,peak_region_fragments,peak_region_cutsites
NO_BARCODE,8692958,1673643,1625,1220361,1114210,0,14527,4668592,0,0,0,0,0,0,0,0,0,0
AAACGAAAGAAAGCAG-1,4,1,0,0,0,0,0,3,0,3,0,0,0,0,0,0,2,4
AAACGAAAGAAAGGGT-1,1,0,0,0,0,0,0,1,0,3,0,0,0,0,0,0,1,2
AAACGAAAGAAATACC-1,3,0,0,0,0,0,0,3,0,3,0,0,0,0,0,0,2,3
AAACGAAAGAAATGGG-1,1200,644,0,109,238,0,6,203,0,0,15,0,0,0,15,0,39,76
AAACGAAAGAAATTCG-1,316,184,0,21,50,0,0,61,0,0,4,0,0,0,4,0,13,25
AAACGAAAGAACAGGA-1,1,0,0,0,0,0,0,1,0,3,0,0,0,0,0,0,0,0
AAACGAAAGAACCCGA-1,1,0,0,1,0,0,0,0,0,0,0,0,0,0,0,0,0,0
AAACGAAAGAACGACC-1,3,0,0,1,0,0,0,2,0,0,1,0,0,0,1,0,2,4

各指标含义：

umn	Type	Description
`barcode`	key	barcodes present in input data
`total`	sequencing	total read-pairs
`duplicate`	mapping	number of duplicate read-pairs
`chimeric`	mapping	number of chimerically mapped read-pairs
`unmapped`	mapping	number of read-pairs with at least one end not mapped
`lowmapq`	mapping	number of read-pairs with <30 mapq on at least one end
`mitochondrial`	mapping	number of read-pairs mapping to mitochondria and non-nuclear contigs
`nonprimary`	mapping	the number of reads that map to non-primary contigs
`passed_filters`	mapping	number of non-duplicate, usable read-pairs i.e. "fragments"
`is_cell_barcode`	cell calling	binary indicator of whether barcode is associated with a cell
`excluded_reason`	cell calling	0: barcode was not excluded; 1: barcode was excluded because it is a gel bead doublet; 2: barcode was excluded because it is low-targeting; 3: barcode was excluded because it is a barcode multiplet
`TSS_fragments`	targeting	number of fragments overlapping with TSS regions
`DNase_sensitive_region_fragments`	targeting	number of fragments overlapping with DNase sensitive regions
`enhancer_region_fragments`	targeting	number of fragments overlapping enhancer regions
`promoter_region_fragments`	targeting	number of fragments overlapping promoter regions
`on_target_fragments`	targeting	number of fragments overlapping any of TSS, enhancer, promoter and DNase hypersensitivity sites (counted with multiplicity)
`blacklist_region_fragments`	targeting	number of fragments overlapping blacklisted regions
`peak_region_fragments`	denovo targeting	number of fragments overlapping peaks
`peak_region_cutsites`	denovo targeting	number of ends of fragments in peak regions

4. BAM 和 .BAM.BAI 文件

根据比对位置排序后的bam文件，其格式与标准的sam/bam文件略有区别，详见之前的文章

Cell Ranger count (gene expression) 输出文件解读_韩建刚（CAAS-UCD）的博客-CSDN博客

5. fragment file，包括fragments.tsv.gz 和 fragments.tsv.gz.tbi

fragments.tsv.gz 文件中行：不同的fragment，列：如下5种属性，

Name	Description
chrom	Reference genome chromosome of fragment
chromStart	Adjusted start position of fragment on chromosome.
chromEnd	Adjusted end position of fragment on chromosome. The end position is exclusive, so represents the position immediately following the fragment interval.
barcode	The 10x cell barcode of this fragment. This corresponds to the CB tag attached to the corresponding BAM file records for this fragment.
readSupport	The total number of read pairs associated with this fragment. This includes the read pair marked unique and all duplicate read pairs.


tail fragments.tsv.gz 
19	55423327	55423435	GATTAGCTCAAGAGAT-1	1
19	55423327	55423435	TCAAGACCATGCGCTG-1	11
19	55423327	55423448	ACGTGGCCAGGTTATC-1	1
19	55423327	55423448	TCAGGTACAGGGCTTC-1	5
19	55423327	55423453	CCTGCTAAGCGTCTGC-1	13
19	55423327	55423453	GGTCATATCAGTGGTT-1	5
19	55423327	55423457	ACCGGGTTCGGGACAA-1	4
19	55423327	55423457	AGTTACGTCGCAACTA-1	2
19	55423327	55423458	GACCGACGTTACGGAG-1	6

6. peaks file, peaks.bed

每一个peak用基因组一段序列区域来表示，其起点-终点分别代表一个酶切事件

Column Number	Name	Description
1	chrom	Reference genome chromosome of peak
2	chromStart	Start position of peak on chromosome.
3	chromEnd	End position of peak on chromosome. The end position is exclusive, so represents the position immediately following the peak interval.

7. peaks 注释

7.1 注释策略：（1）一个peak 可以被比对到多个基因；（2）一个peak 只能是promoter peak 或distal peak的一种；（3）只有蛋白编码基因能够被注释到。

7.2 注释具体过程：（1）如果peak在启动子区域（TSS位点 -1000bp，+100bp），会被注释为promoter peak；（2）如果在TSS 200kb 以内，但没有被注释成 promoter peak，则会被注释为 distal peak；（3）如果一个peak位于转录本中（基因内），同时既不是promoter peak 也不是distal peak，则会被定义为distal peak，但距离会被设为0；（4）如果一个 peak 在上边三步没有被注释到任何基因，最终会被定义为 intergenic peak

7.3 peak_annotation.tsv 格式

共包含6列，前三列为 peak 染色体位置，第四列为注释基因名字，第五列为peak到基因的距离，正值表示到 peak 起点位于 TSS 下游，负值表示 peak 终点位于 TSS 上游，0 表示 peak 与TSS 重叠或者位于基因转录本区域。

Name	Description
chrom	Contig that contains the peak
start	Peak start location
end	Peak end location
gene	Gene symbol based on the gene annotation in the reference.
distance	Distance of peak from TSS of gene. Positive distance means the start of the peak is downstream of the position of the TSS, whereas negative distance means the end of the peak is upstream of the TSS. Zero distance means the peak overlaps with the TSS or the peak overlaps with the transcript body of the gene.
peak_type	Can be "promoter", "distal" or "intergenic".


head peak_annotation.tsv 
chrom	start	end	gene	distance	peak_type
1	12116	12985	ENSOARG00020000038	-13113	distal
1	29918	30788	ENSOARG00020000038	3821	distal
1	34037	35000	ENSOARG00020000038	7940	distal
1	36768	37257	ENSOARG00020000038	10671	distal
1	37368	38200	ENSOARG00020000038	11271	distal
1	46853	47720	ENSOARG00020000038	20756	distal
1	49556	50414	ENSOARG00020000038	23459	distal
1	59206	60122	FAM240C	-27312	distal
1	63961	64830	FAM240C	-22604	distal