Users may refer to this section as a format for testing SAW process, of the files show in this chapter as the reference in testing SAW pipelines. This chapter includes the statistics results and examples of critical files for each key step.
SN：SS200000135TL_D1
"…" in the demo stands for some lines of log information that can be omitted.
1. mapping
1.1 Statistical Report for CID Mapping and Filtering
$ cat /path/to/output/01.mapping/E100026571_L01_trim_read_1_barcodeMap.stat ...
getBarcodePositionMap_uniqBarcodeTypes: 645784920
total_reads: 1002214171
reads_with_polyA: 131113905 13.08%
reads_filteredByPolyA: 22008148 2.20%
mapped_reads: 826344259 82.45%
reads_with_adapter: 9007116 0.90%
reads_with_dnb: 42264284 4.22%
barcode_exactlyOverlap_reads: 682746301 68.12%
barcode_misOverlap_reads: 143590127 14.33%
barcode_withN_reads: 7831 0.00%
reads_with_rRNA: 0 0.00%
Q10_bases_in_barcode: 99.54%
Q20_bases_in_barcode: 97.49%
Q30_bases_in_barcode: 91.74%
Q10_bases_in_umi: 99.26%
Q20_bases_in_umi: 96.32%
Q30_bases_in_umi: 89.45%
Q10_bases_in_seq: 99.47%
Q20_bases_in_seq: 97.12%
Q30_bases_in_seq: 91.08%
umi_filter_reads: 8265089 0.82%
umi_with_N_reads: 13025 0.00%
umi_with_polyA_reads: 12365 0.00%
umi_with_low_quality_base_reads: 8239699 0.82%
mapped_dnbs: 75619113
...
1.2 Statistical Report for Reference Genome Alignment
$ cat /path/to/output/01.mapping/E100026571_L01_trim_read_1.Log.final.out
...
Number of input reads | 769052654
Average input read length | 95
UNIQUE READS:
Uniquely mapped reads number | 645319822
Uniquely mapped reads % | 83.91%
Average mapped length | 95.14
Number of splices: Total | 67634124
Number of splices: Annotated (sjdb) | 65711163
Number of splices: GT/AG | 66445242
Number of splices: GC/AG | 457783
Number of splices: AT/AC | 41573
Number of splices: Non-canonical | 689526
Mismatch rate per base, % | 0.50%
Deletion rate per base | 0.07%
Deletion average length | 3.91
Insertion rate per base | 0.03%
Insertion average length | 1.25
MULTI-MAPPING READS:
Number of reads mapped to multiple loci | 87828411
% of reads mapped to multiple loci | 11.42%
Number of reads mapped to too many loci | 5333051
% of reds mapped to too many loci | 0.69%
UNMAPPED READS:
Number of reads unmapped: too many mismatches | 0
% of reads unmapped: too many mismatches | 0.00%
Number of reads unmapped: too short | 29365488
% of reads unmapped: too short | 3.82%
Number of reads unmapped: other | 1205882
% of reads unmapped: other | 0.16%
CHIMERIC READS:
Number of chimeric reads | 0
% of chimeric reads | 0.00%
1.3 Example of mapping BAM
$ samtools view /path/to/output/01.mapping/E100026571_L01_trim_read_1.Aligned.sortedByCoord.out.bam | head -2
E100026571L1C007R00303973559 256 1 3000644 3 100M * 0 0 GCCTCATTGTGCCCCATATGTTTGCCTATGTTGTGGACTTATTTTCATTAAACTTTAAAACATCTTTAATTTTTTTCTTTATTTCATCATTGACCAAGCT -FCA9D?GFFD<-D<cgfegd-dg*fgfdfbe;e(9bgge38fffg9gg;0?ggfgb?e@g:ggg3gf79f0ggdg?g
2. merge
2.1 Example of Mapped CID List with Reads Count File
$ head /path/to/output/02.merge/SS200000135TL_D1.merge.barcodeReadsCount.txt
7127 18002 48
4348 19028 1
14130 8635 1
7618 14537 24
4912 10945 5
16783 12914 1
15539 8177 1
9288 8082 14
7274 16533 59
9087 10657 10
3. count
3.1 Statistical Report for MID Filtering and Gene Annotation
$ cat /path/to/output/03.count/SS200000135TL_D1.Aligned.sortedByCoord.out.merge.q10.dedup.target.bam.summary.stat
## FILTER & DEDUPLICATION METRICS
TOTAL_READS PASS_FILTER ANNOTATED_READS UNIQUE_READS FAIL_FILTER_RATE FAIL_ANNOTATE_RATE DUPLICATION_RATE
733148233 645319822 533743961 108363128 11.98 17.29 79.70
## ANNOTATION METRICS
TOTAL_READS MAP EXONIC INTRONIC INTERGENIC TRANSCRIPTOME ANTISENSE
645319822 645319822 484480416 49263545 111575861 533743961 110185975
100.0 100.0 75.1 7.6 17.3 82.7 17.1
3.2 Example of Annotated BAM
$ samtools view /path/to/output/03.count/SS200000135TL_D1.Aligned.sortedByCoord.out.merge.q10.dedup.target.bam | head -2
E100026571L1C007R00303973559 768 1 3000644 3 100M * 0 0 GCCTCATTGTGCCCCATATGTTTGCCTATGTTGTGGACTTATTTTCATTAAACTTTAAAACATCTTTAATTTTTTTCTTTATTTCATCATTGACCAAGCT -FCA9D?GFFD<-D<CGFEGD-DG*FGFDFBE;E(9BGGE38FFFG9GG;0?GGFGB?E@G:GGG3GF79F0GGDG?G<D>F;EG,G?<<CD4>G=>B+C NH:i:2 HI:i:1 AS:i:94 nM:i:2 Cx:i:8839 Cy:i:7539 UR:Z:120CF
E100026571L1C003R03702347721 0 1 3001778 255 100M * 0 0 GTATGACATCTGTCCAGGATCTTCTAGCTTTCATAGTCTCTGGTGAGAAGTCTGGAGTAATTCTAATAGGCCTGCATTTATATGTTACTTGACCTTTTTC EEFEDFFEFFFFEFFFFEC@EFFFFDFFEEFFEFFFFCFCEFFAFBFCED??FGBEFFDC:FFFDCFAF4FAFFDFFDG?DFBD.F@FECA/FEDEFFAA NH:i:1 HI:i:1 AS:i:92 nM:i:3 Cx:i:12136 Cy:i:14034 UR:Z:C0808 XF:i:2
3.3 Example of count Gene Expression Matrix
$ h5dump -n /path/to/output/03.count/SS200000135TL_D1.raw.gef
HDF5 "/path/to/output/03.count/SS200000135TL_D1.raw.gef" {
FILE_CONTENTS {
group /
group /geneExp
group /geneExp/bin1
dataset /geneExp/bin1/exon
dataset /geneExp/bin1/expression
dataset /geneExp/bin1/gene
}
}
$ h5dump -d /geneExp/bin1/expression /path/to/output/03.count/SS200000135TL_D1.raw.gef | head -15
HDF5 "/path/to/output/03.count/SS200000135TL_D1.raw.gef" {
DATASET "/geneExp/bin1/expression" {
DATATYPE H5T_COMPOUND {
H5T_STD_U32LE "x";
H5T_STD_U32LE "y";
H5T_STD_U8LE "count";
}
DATASPACE SIMPLE { ( 76210618 ) / ( 76210618 ) }
DATA {
(0): {
636,
12671,
2
},
(1): {
$ h5dump -d /geneExp/bin1/gene /path/to/output/03.count/SS200000135TL_D1.raw.gef | head -20
HDF5 "/path/to/output/03.count/SS200000135TL_D1.raw.gef" {
DATASET "/geneExp/bin1/gene" {
DATATYPE H5T_COMPOUND {
H5T_STRING {
STRSIZE 32;
STRPAD H5T_STR_NULLTERM;
CSET H5T_CSET_ASCII;
CTYPE H5T_C_S1;
} "gene";
H5T_STD_U32LE "offset";
H5T_STD_U32LE "count";
}
DATASPACE SIMPLE { ( 24670 ) / ( 24670 ) }
DATA {
(0): {
"0610005C13Rik",
0,
45
},
(1): {3.4 Example of count Sampling File
$ head -8 /path/to/output/03.count/SS200000135TL_D1_raw_barcode_gene_exp.txt
x y geneIndex MIDIndex readCount
9602 7705 10551 611723 2
4888 10392 10551 665954 4
8901 7096 10551 881671 1
8901 7096 10551 357383 20
7397 18783 10551 355789 1
9155 13032 10551 297666 1
9155 13032 10551 298690 1
4. register and imageTools
4.1 Registered Image
File /path/to/output/04.register/ssDNA_fov_stitched_transformed.tif and /path/to/output/04.register/ssDNA_SS200000135TL_D1_regist.tif.
/path/to/output/04.register/ssDNA_fov_stitched_transformed.tif
/path/to/output/04.register/ssDNA_SS200000135TL_D1_regist.tif
4.2 Image Process Record File
$ h5dump -n /path/to/output/04.register/SS200000135TL_D1_20220527_201353_1.1.0.ipr
HDF5 "/path/to/output/04.register/SS200000135TL_D1_20220527_201353_1.1.0.ipr" {
FILE_CONTENTS {
group /
group /ManualState
dataset /Preview
group /StereoResepSwitch
group /ssDNA
group /ssDNA/CellSeg
dataset /ssDNA/CellSeg/CellMask
dataset /ssDNA/CellSeg/CellSegTrace
group /ssDNA/ImageInfo
dataset /ssDNA/ImageInfo/RGBScale
group /ssDNA/QCInfo
group /ssDNA/QCInfo/CrossPoints
dataset /ssDNA/QCInfo/CrossPoints/0_0
...
dataset /ssDNA/QCInfo/CrossPoints/9_8
group /ssDNA/Register
dataset /ssDNA/Register/MatrixTemplate
group /ssDNA/Stitch
group /ssDNA/Stitch/ScopeStitch
dataset /ssDNA/Stitch/ScopeStitch/GlobalLoc
dataset /ssDNA/Stitch/ScopeStitch/ScopeHorizontalJitter
dataset /ssDNA/Stitch/ScopeStitch/ScopeJitterDiff
dataset /ssDNA/Stitch/ScopeStitch/ScopeVerticalJitter
group /ssDNA/Stitch/StitchEval
dataset /ssDNA/Stitch/StitchEval/GlobalDeviation
dataset /ssDNA/Stitch/StitchEval/StitchEvalH
dataset /ssDNA/Stitch/StitchEval/StitchEvalV
dataset /ssDNA/Stitch/TemplatePoint
dataset /ssDNA/Stitch/TransformTemplate
group /ssDNA/TissueSeg
dataset /ssDNA/TissueSeg/TissueMask
}
}
$ h5dump -A /path/to/output/04.register/SS200000135TL_D1_20220527_201353_1.1.0.ipr | head -20
HDF5 "/path/to/output/04.register/SS200000135TL_D1_20220527_201353_1.1.0.ipr" {
GROUP "/" {
ATTRIBUTE "IPRVersion" {
DATATYPE H5T_STRING {
STRSIZE H5T_VARIABLE;
STRPAD H5T_STR_NULLTERM;
CSET H5T_CSET_UTF8;
CTYPE H5T_C_S1;
}
DATASPACE SCALAR
DATA {
(0): "0.2.0"
}
}
GROUP "ManualState" {
ATTRIBUTE "calibration" {
DATATYPE H5T_ENUM {
H5T_STD_I8LE;
"FALSE" 0;
"TRUE" 1;4.3 ImageTools merge
Merged image of microscopy image ssDNA_SS200000135TL_D1_regist.tif and tissue segmentation mask file ssDNA_SS200000135TL_D1_tissue_cut.tif to check tissue segmentation performance.
Part of merged image of microscopy image ssDNA_SS200000135TL_D1_regist.tif and cell segmentation mask file ssDNA_SS200000135TL_D1_mask.tif to check cell segmentation performance.
4.4 ImageTools overlay
Stack stitching template onto the ssDNA_fov_stitched_transformed.tif to check the result of stitching.
Stack registration template onto the ssDNA_SS200000135TL_D1_regist.tif to check the result of registration.
5. tissueCut
5.1 Statistical Report for Tissue Covered Region
$ cat /path/to/output/05.tissuecut/tissuecut.stat
# Tissue Statistic Analysis with Stain Image
Contour_area: 87086375
Number_of_DNB_under_tissue: 36521212
Ratio: 41.94%Total_gene_type: 24289
MID_counts: 89679129
Fraction_MID_in_spots_under_tissue: 82.76%
Reads_under_tissue: 709807297
Fraction_reads_in_spots_under_tissue: 83.99%
binSize=1
Mean_reads_per_spot: 15.10
Median_reads_per_spot: 9.00
Mean_gene_type_per_spot: 1.71
Median_gene_type_per_spot: 1
Mean_Umi_per_spot: 2.46
Median_Umi_per_spot: 2
binSize=20
Mean_reads_per_spot: 3241.11
Median_reads_per_spot: 2782.00
Mean_gene_type_per_spot: 241.08
Median_gene_type_per_spot: 227
Mean_Umi_per_spot: 409.56
Median_Umi_per_spot: 370
binSize=50
Mean_reads_per_spot: 20054.45
Median_reads_per_spot: 18285.00
Mean_gene_type_per_spot: 1165.56
Median_gene_type_per_spot: 1133
Mean_Umi_per_spot: 2534.10
Median_Umi_per_spot: 2346
binSize=100
Mean_reads_per_spot: 78867.48
Median_reads_per_spot: 72545.00
Mean_gene_type_per_spot: 3110.83
Median_gene_type_per_spot: 3117
Mean_Umi_per_spot: 9964.35
Median_Umi_per_spot: 9205
binSize=150
Mean_reads_per_spot: 174614.34
Median_reads_per_spot: 162073.00
Mean_gene_type_per_spot: 4926.51
Median_gene_type_per_spot: 5065
Mean_Umi_per_spot: 22066.71
Median_Umi_per_spot: 20430
binSize=200
Mean_reads_per_spot: 305687.91
Median_reads_per_spot: 285723.00
Mean_gene_type_per_spot: 6424.38
Median_gene_type_per_spot: 6747
Mean_Umi_per_spot: 38621.50
Median_Umi_per_spot: 36060
5.2 Example of Gene Expression Matrix for Tissue Covered Region
$ h5dump -n /path/to/output/05.tissuecut/SS200000135TL_D1.tissue.gef
HDF5 "/path/to/output/05.tissuecut/SS200000135TL_D1.tissue.gef" {
FILE_CONTENTS {
group /
group /geneExp
group /geneExp/bin1
dataset /geneExp/bin1/exon
dataset /geneExp/bin1/expression
dataset /geneExp/bin1/gene
}
}
$ h5dump -d /geneExp/bin1/expression /path/to/output/05.tissuecut/SS200000135TL_D1.tissue.gef | head -15
HDF5 "/path/to/output/05.tissuecut/SS200000135TL_D1.tissue.gef" {
DATASET "/geneExp/bin1/expression" {
DATATYPE H5T_COMPOUND {
H5T_STD_U32LE "x";
H5T_STD_U32LE "y";
H5T_STD_U8LE "count";
}
DATASPACE SIMPLE { ( 62542665 ) / ( 62542665 ) }
DATA {
(0): {
6148,
10906,
1
},
(1): {
h5dump -d /geneExp/bin1/gene /path/to/output/05.tissuecut/SS200000135TL_D1.tissue.gef | head -20
HDF5 "/path/to/output/05.tissuecut/SS200000135TL_D1.tissue.gef" {
DATASET "/geneExp/bin1/gene" {
DATATYPE H5T_COMPOUND {
H5T_STRING {
STRSIZE 64;
STRPAD H5T_STR_NULLPAD;
CSET H5T_CSET_ASCII;
CTYPE H5T_C_S1;
} "gene";
H5T_STD_U32LE "offset";
H5T_STD_U32LE "count";
}
DATASPACE SIMPLE { ( 24289 ) / ( 24289 ) }
DATA {
(0): {
"0610005C13Rik",
0,
24
},
(1): {5.3 Example of Gene Expression Matrix for a complete GEF
$ h5dump -n /path/to/output/03.count/SS200000135TL_D1.gef
HDF5 "/path/to/output/03.count/SS200000135TL_D1.gef" {
FILE_CONTENTS {
group /
group /geneExp
group /geneExp/bin1
dataset /geneExp/bin1/exon
dataset /geneExp/bin1/expression
dataset /geneExp/bin1/gene
group /geneExp/bin10
dataset /geneExp/bin10/exon
dataset /geneExp/bin10/expression
dataset /geneExp/bin10/gene
group /geneExp/bin100
dataset /geneExp/bin100/exon
dataset /geneExp/bin100/expression
dataset /geneExp/bin100/gene
group /geneExp/bin20
dataset /geneExp/bin20/exon
dataset /geneExp/bin20/expression
dataset /geneExp/bin20/gene
group /geneExp/bin200
dataset /geneExp/bin200/exon
dataset /geneExp/bin200/expression
dataset /geneExp/bin200/gene
group /geneExp/bin50
dataset /geneExp/bin50/exon
dataset /geneExp/bin50/expression
dataset /geneExp/bin50/gene
group /geneExp/bin500
dataset /geneExp/bin500/exon
dataset /geneExp/bin500/expression
dataset /geneExp/bin500/gene
group /stat
dataset /stat/gene
group /wholeExp
dataset /wholeExp/bin1
dataset /wholeExp/bin10
dataset /wholeExp/bin100
dataset /wholeExp/bin20
dataset /wholeExp/bin200
dataset /wholeExp/bin50
dataset /wholeExp/bin500
group /wholeExpExon
dataset /wholeExpExon/bin1
dataset /wholeExpExon/bin10
dataset /wholeExpExon/bin100
dataset /wholeExpExon/bin20
dataset /wholeExpExon/bin200
dataset /wholeExpExon/bin50
dataset /wholeExpExon/bin500
}
}
$ h5dump -d /stat/gene /path/to/output/03.count/SS200000135TL_D1.gef | head -20
HDF5 "/path/to/output/03.count/SS200000135TL_D1.gef" {
DATASET "/stat/gene" {
DATATYPE H5T_COMPOUND {
H5T_STRING {
STRSIZE 64;
STRPAD H5T_STR_NULLTERM;
CSET H5T_CSET_ASCII;
CTYPE H5T_C_S1;
} "gene";
H5T_STD_U32LE "MIDcount";
H5T_IEEE_F32LE "E10";
}
DATASPACE SIMPLE { ( 24670 ) / ( 24670 ) }
DATA {
(0): {
"Gm42418",
5860952,
60.1028
},
(1): {6. cellCut
6.1 Example of Gene Expression Matrix for Cell Bins
$ h5dump -n /path/to/output/051.cellcut/SS200000135TL_D1.cellbin.gef
HDF5 "/path/to/output/051.cellcut/SS200000135TL_D1.cellbin.gef" {
FILE_CONTENTS {
group /
group /cellBin
dataset /cellBin/blockIndex
dataset /cellBin/blockSize
dataset /cellBin/cell
dataset /cellBin/cellBorder
dataset /cellBin/cellExon
dataset /cellBin/cellExp
dataset /cellBin/cellExpExon
dataset /cellBin/cellTypeList
dataset /cellBin/gene
dataset /cellBin/geneExon
dataset /cellBin/geneExp
dataset /cellBin/geneExpExon
}
}7. saturation
$ cat /path/to/output/07.saturation/sequence_saturation.tsv
sample bar_x bar_y1 bar_y2 bar_umi bin_x bin_y1 bin_y2 bin_umi
0.05 26687198 0.250475 1 20002730 26687198 0.273744 3030 7041
0.1 53374396 0.389862 1 32565765 53374396 0.409615 4045 11464
0.2 106748792 0.542456 1 48842313 106748792 0.557064 4962 17194
0.3 160123200 0.625182 1 60017028 160123200 0.636707 5435 21128
0.4 213497584 0.677501 1 68852648 213497584 0.6871 5778 24238
0.5 266871984 0.713814 1 76374913 266871984 0.722081 6052 26886
0.6 320246400 0.740741 1 83026752 320246400 0.748035 6234 29228
0.7 373620768 0.761599 1 89071589 373620768 0.768155 6381 31356
0.8 426995168 0.778242 1 94689701 426995168 0.784222 6528 33334
0.9 480369568 0.79188 1 99974373 480369568 0.797395 6639 35194
1 533743961 0.803326 1 104973406 533743961 0.808462 6718 36641
8. report
8.1 Example of Statistical Summary Report
$ head /path/to/output/08.report/SS200000135TL_D1.statistics.json
{
"version": "version_v2",
"1.Filter_and_Map": {
"1.1.Adapter_Filter": [
{
"Sample_id": "E100026571_L01_trim_read_1",
"getCIDPositionMap_uniqCIDTypes": "645784920",
"total_reads": "1002214171",
"CID_withN_reads": "8031 (0.00 %)",
"mapped_reads": "845113606 (84.32 %)",8.2 HTML Report
