Appendices and References

SAW operation manual

Appendix A: Recommended Directory Structure for Raw Data

Recommend to organize raw data as below if users prefer to use SAW shell script provided on the SAW GitHub page (https://github.com/STOmics/SAW).

$ tree
    .
    |-- image
    |   |-- SS200000135TL_D1_20220527_201353_1.1.0.ipr
    |   `-- SS200000135TL_D1_20220527_201353_1.1.0.tar.gz
    |-- mask
    |   `-- SS200000135TL_D1.barcodeToPos.h5
    |-- md5
    |-- reads
    |   |-- E100026571_L01_trim_read_1.fq.gz
    |   `-- E100026571_L01_trim_read_2.fq.gz
    `-- reference
        |-- STAR_SJ100
        |   |-- Genome
        |   |-- SA
        |   |-- SAindex
        |   |-- chrLength.txt
        |   |-- chrName.txt
        |   |-- chrNameLength.txt
        |   |-- chrStart.txt
        |   |-- exonGeTrInfo.tab
        |   |-- exonInfo.tab
        |   |-- geneInfo.tab
        |   |-- genomeParameters.txt
        |   |-- genomeParameters_bkp.txt
        |   |-- sjdbInfo.txt
        |   |-- sjdbList.fromGTF.out.tab
        |   |-- sjdbList.out.tab
        |   `-- transcriptInfo.tab
        |-- genes.gtf
        `-- genome.fa
     
    5 directories, 24 files

Appendix B: SAW ST Output File List

splitMask	*.SN.barcodeToPos.bin	Split Stereo-seq Chip T mask file according to the CID.
mapping	lane.Aligned.sortedByCoord.out.bam	Binary Alignment/Map file, used for storing the sequence alignment information.
	lane.barcodeReadsCount.txt	BMapped CID list file with reads counts for each CID, the three columns record x, y, and read count.
	lane.Log.final.out	Summary mapping statistics after mapping job is complete (STAR output).
	lane.Log.out	Main log file in STAR mapping (STAR output).
	lane.Log.progress.out	Reports job process statistics (STAR output).
	lane.SJ.out.tab	Splice junctions detected in the mapping (STAR output).
	lane.bcPara	A parameters file defines CID mapping options.
	lane_barcodeMap.stat	Statistical report of CID mapping, such as CID mapped reads count, reads sequencing quality, mapped DNB count, etc.
merge	SN.merge.barcodeReadsCount.txt	Mapped CID list file with reads counts for each CID., the three columns record x, y, and read count.
count	SN.Aligned.sortedByCoord.out.merge. q10.dedup.target.bam	Annotated BAM file sorted by coordinate, includes uniquely mapped reads and multi-mapped reads whose HI:i tag is 1.
	SN.Aligned.sortedByCoord.out.merge. q10.dedup.target.bam.csi	Index file of annotated BAM.
	SN.Aligned.sortedByCoord.out.merge. q10.dedup.target.bam.summary.stat	Statistical summary report file for count. Total reads in filter&deduplication metrics stand for the total alignment record count in BAM. Pass filter and total reads in annotation metrics are the same, they represent uniquely mapped reads that will be used for annotation, MID correction and quantification.
	SN.raw.gef	Gene expression file in hdf5 format. This is the first raw matrix that includes the expression information over a complete chip region. It only includes geneExp group for the bin size of 1. The origin of expression matrix has been calibrated to (0,0), and the offset x and y has been record as minX and minY in the attribute of geneExp/expression dataset.
	SN_raw_barcode_gene_exp.txt	A space-separate file records coordinate, gene, MID, and count information. Which is prepared to be a sampling file that performs sequence saturation. The 5 columns are y, x, geneIndex, MIDIndex, readCount.
register & imageTools ipr2img	date-hh-mm-ss.log	Log file.
	SN or other user specified name for the image folder used when input into imageQC/imageStudio	This subdirectory stores raw small image pieces in TIFF format.
	SN_0000_0000_YYYY-MM-DD_hh-mm-ss-n.tif	Small image pieces in TIFF format.
	fov_stitched_transformed.tif	The stitched panoramic image that has pre-registered with the track line pattern template. So that it will not need to further rotate a non-right angle or scale.
		The stitched panoramic image from DAPI/IF layers that has pre-registered with the track line pattern template. So that it will not need to further rotate a non-right angle or scale.
		Track line cross point template for the registered DAPI/IF image. Used for assessing registration result.
	SN_	IPR format image process record file which records basic image information gathered from ImageQC/ImageStudio.
		Registered cell segmentation binary image file from DAPI/IF layers in TIFF format.
		Registered panoramic image file from DAPI/IF layers in TIFF format.
	SN.rpi	Registered panoramic image file, tissue area boundary, and cell boundaries (downsampled) that stores as an image pyramid.
	SN_tissue_bbox.csv	Registered panoramic image size.
		The tissue segmentation result of registered panoramic image file from DAPI/IF layers in TIFF format.
		Track line cross point template for the
tissueCut	SN.gef	A gene expression file in hdf5 format. This file is a complete GEF format which includes geneExp group and wholeExp group in bin1, 10, 20, 50, 100, 200, and 500. It also includes a stat group. The origin of expression matrix has been calibrated to (0,0), and the offset x and y has been recorded as minX and minY in the attribute of geneExp/expression dataset.
	SN.tissue.gef	A gene expression file in hdf5 format. Tissue GEF includes the expression information of the tissue coverage region. It only includes geneExp group for the bin size of 1. The origin of expression matrix has been calibrated to (0,0), and the offset x and y has been recorded as minX and minY in the attribute of geneExp/expression dataset same to raw GEF.
	SN.gem.gz	The compressed gene expression matrix that stores genes spatial expression data.
	SN.tissue.gem.gz	The compressed gene expression matrix that stores genes spatial expression data in the tissue region.
	tissue_fig	This directory stores the statistical plots for the tissue coverage region
	scatter_100x100_MID_gene_counts.png	Scatter plot of MID count and gene number in each bin (bin100)
	scatter_150x150_MID_gene_counts.png	Scatter plot of MID count and gene number in each bin (bin150)
	scatter_200x200_MID_gene_counts.png	Scatter plot of MID count and gene number in each bin (bin200)
	scatter_20x20_MID_gene_counts.png	Scatter plot of MID count and gene number in each bin (bin20)
	scatter_50x50_MID_gene_counts.png	Scatter plot of MID count and gene number in each bin (bin50)
	statistic_100x100_MID_gene_DNB.png	Univariate distribution of MID count, gene numbers, and DNB numbers with rug along the x axis (bin100)
	statistic_150x150_MID_gene_DNB.png	Univariate distribution of MID count, gene numbers, and DNB numbers with rug along the x axis (bin150)
	statistic_200x200_MID_gene_DNB.png	Univariate distribution of MID count, gene numbers, and DNB numbers with rug along the x axis (bin200)
	statistic_20x20_MID_gene_DNB.png	Univariate distribution of MID count, gene numbers, and DNB numbers with rug along the x axis (bin20)
	statistic_50x50_MID_gene_DNB.png	Univariate distribution of MID count, gene numbers, and DNB numbers with rug along the x axis (bin50)
	violin_100x100_MID_gene.png	Violin plots show the distribution of deduplicated MID count and gene number in each bin (bin100)
	violin_150x150_MID_gene.png	Violin plots show the distribution of deduplicated MID count and gene number in each bin (bin150)
	violin_200x200_MID_gene.png	Violin plots show the distribution of deduplicated MID count and gene number in each bin (bin200)
	violin_20x20_MID_gene.png	Violin plots show the distribution of deduplicated MID count and gene number in each bin (bin20)
	violin_50x50_MID_gene.png	Violin plots show the distribution of deduplicated MID count and gene number in each bin (bin50)
	tissuecut.stat	Statistical report for tissue coverage region.
cellCut	SN.cellbin.gef	A gene expression file for cells in hdf5 format. Cellbin GEF includes the expression information of the cells, such as the coordinate of the centroid, boundary coordinates, expression of genes, and cell area. The cell is record by its boundary. The origin of expression matrix has been calibrated to (0,0), and the coordinate offset x and y has been record as offsetX and offsetY in the attribute of the GEF file same to minX and minY in the raw GEF file.
spatialCluster	SN.spatial.cluster.h5ad	H5AD file for spatial clustering analysis result.
cellCluster	SN.cell.cluster.h5ad	H5AD file for cell clustering analysis result.
saturation	plot_1x1_saturation.png	Sequencing saturation analysis plots for bin1. Calculated by 1- (unique reads/total reads) for each bin (bin1).
	plot_200x200_saturation.png	Sequencing saturation analysis plots for bin200. Calculated by 1- (unique reads/total reads) for each bin (bin1).
	sequence_saturation.tsv	Sequence saturation file. The nine colums are sampling component (#sample), bin1 total reads (bar_x), sequence saturation value at bin1 (bar_y1), median gene count at bin1 (bar_y2), unique reads at bin1 (bar_umi), bin200 total reads (bin_x), sequence saturation value at bin200 (bin_y1), median gene count at bin200 (bin_y2), and unique reads at bin200 (bin_umi), respectively.
report	SN.report.html	HTML analytical report.
	SN.statistics.json	Statistical summary report in JSON format. It gathers all important statistical metrics from the statistical report in each step.
	scatter_1x1_MID_gene_counts.png	Scatter plot of MID count and gene number in each cell
	statistic_1x1_MID_gene_DNB.png	Univariate distribution of MID count, gene numbers, and DNB numbers with rug along the x axis of each cell
	violin_1x1_MID_gene.png	Violin plots show the distribution of deduplicated MID count and gene number in each cell

Appendix C: Handling Errors and Exceptions Common Errors

Common Errors

File access error:
Some examples of file access error:

terminate called after throwing an instance of 'std::invalid_argument'
    what():  Could not open the file: xxxxxx
    ...

#006: H5FDsec2.c line 352 in H5FD__sec2_open(): unable to open file: name = '/path/to/hdf5/file', errno = 2, error message = 'No such file or directory', flags = 0, o_flags = 0
        major: File accessibility
        minor: Unable to open file
    ...

Solution:

# Attempt 1: Bind file path before execute command.
    $ export SINGULARITY_BIND="/path/to/file/directory"
    
    # Attempt 2: Turn off the HDF5 lock on the file by running the command below before running SAW.
    $ export HDF5_USE_FILE_LOCKING=FALSE

mapping

readNameSeparator error:

EXITING: FATAL INPUT ERROR: empty value for parameter “readNameSeparator” in input “Command-Line”
    SOLUTION: use non-empty value for this parameter

Solution: try to delete --readNameSeparator \” \” from the command

limitBAMsortRAM error:

Error code: SAW-A10183
    
    EXITING because of fatal ERROR: not enough memory for BAM sorting:
    SOLUTION: re-run STAR with at least –limitBAMsortRAM xxxxxxxxx

Solution: Modify the value specified by the --limitBAMsortRAM refer to the SOLUTION in the stderr.

--limitOutSJcollapsed error:

EXITING because of fatal error: buffer size of SJ output is too small
    Solution: increase input parameter --limitOutSJcollapsed

Solution: the parameters --limitOutSJcollapsed and --limitIObufferSize are paired. Please replace them with --limitOutSJcollapsed 10000000 --limitIObufferSize=480000000 and try again.

imageTools

OSError raises for imageTools merge

OSError: [Errno 30] Read-only file system: '/opt/saw_v5.4.0_software/pipeline/imageTools/imagetools-1.0.0/log'

Solution: Error raises because the -o input is a filename without path. Try to input /path/to/output/file.rpi or ./file.rpi for -o.

Appendix D: Error Codes

SAW error codes are designed to catch error information and describe it in an easily understood way. Users can use these messages to fix the problem by themselves. The error messages will be output to the "errcode.log" file.

The error code is designed to express in three parts, date-time, error code, and explanation. The date-time information could help users differentiate errors in different program executions. The error code part provides information for the pipeline modules and error types. The explanation section displays the most explicit message stating the error/exception and possible solutions.

splitMask	SAW-A00001	Parameters invalid or missing	e.g. "parameters error"Please check your input parameters. Some required parameters might be missed. e.g. "splitBcPos error, expected 1_24 or 2_25" Please check your input CID position is either 1_24 or 2_25.
	SAW-A00002	File open failed	Please check the input file exists and has the correct access permission.
	SAW-A00003	File parse failed	e.g. "only support .bin or .h5 file." Please check your input file is in the correct file format.
	SAW-A00004	File open failed	e.g. "cannot write to file, /path/to/file" Please check your output directory path is an existing path.
	SAW-A00005	Other API error	Please check Appendix C or contact FAS/FBS for help.
	SAW-A00006	Software exception	Please check Appendix C or contact FAS/FBS for help.
CIDCount	SAW-A00021	Parameters invalid or missing	Please check your input parameters. Some required parameters might be missed.
	SAW-A00022	File open failed	e.g. "cannot open such file, /path/to/file" Please check the input file exists and has the correct access permission.
	SAW-A00023	Failed to parser the file	Please check your input file is in the correct file format.
	SAW-A00024	Other API error	Please check Appendix C or contact FAS/FBS for help.
	SAW-A00025	Software exception	Please check Appendix C or contact FAS/FBS for help.
mapping	SAW-A10101	Parameters invalid or missing	e.g. "EXITING: FATAL INPUT ERROR: unrecognized parameter name "outSAMattribute" in input "Command-Line-Initial"" Please check the spelling of your argument and parameters. e.g. "please check the umi position and length" Please check the length of the reads in FQ1 are consistent with the parameters set for barcode length and umi length. For example, the length of the sum of barcode and umi set in bcPara file is 35 bp, but one of the reads in FQ1 is 30 bp, then you will see A10101 error code. Please check the parameters set in the bcPara file are consistent with your FQs.
	SAW-A10102	File open failed	e.g. "Error, cannot open the file which be expected in gz or ascii format" Please check the file permission and file format. e.g. "barcodePositionMapFile does not exists: /path/to/mask" Please check the mask file exists and has the correct access permission.
	SAW-A10103	File parse failed	e.g. "sequence and quality have different length" Please check the completeness of the reads in FQ. This issue may arise if the FQs are in incorrect format or the file was incompletely written or transferred.
	SAW-A10104	Invalid data or data exception	Error data. Please check the file format and content.
	SAW-A10105	File deletion failed	This error arose if "_STARtmp" directory failed to be deleted. Please check whether the program has completely finished according to the .Log.progress.out or _barcodeMap.stat file.
	SAW-A10106	File IO failed	Please contact FAS/FBS for help.
	SAW-A10107	Failure on APIs of system and libraries	Please contact FAS/FBS for help.
	SAW-A10108	Software assert	Please contact FAS/FBS for help.
	SAW-A10109	Software exception	Please contact FAS/FBS for help.
	SAW-A10110	Allocate memory error	This error occurs when you run out of RAM. You may need to kill some jobs that use the same RAM or upgrade your hardware to get more RAM resources for your job.
	SAW-A10111	Out of disk space	This error occurs when you store too many files on your hard disk. Please remove some files to free disk space.
	SAW-A10200	Parameter missing	Please check your input parameters. Some required parameters might be missed.
	SAW-A10201	CID comparison rate is too low	This error usually arises because the CID information of the input FQs is not the same as the CID in the input mask file. Please use the correct SN-FQ pairs for mapping.
	SAW-A10202	Fail to create the index for the BAM file	Please contact FAS/FBS for help.
	SAW-A10300	Fail to load indexed reference	Please check the existence, access permission, and completeness for the indexed reference.
count	SAW-A20001	Parameter missing	Please check your input parameters. Some required parameters might be missed.
	SAW-A20002	File open failed	Please check the input file exists and has the correct access permission.
	SAW-A20003	File parse failed	Please check your input BAM header is in the correct file format.
	SAW-A20004	Allocate memory error	This error occurs when you run out of RAM. You may need to kill some jobs that use the same RAM or upgrade your hardware to get more RAM resources for your job.
	SAW-A20009	Software exception	Please contact FAS/FBS for help.
	SAW-A20101	Parameter missing	Please check your input parameters. Some required parameters might be missed.
	SAW-A20102	File open failed	Please check the file that exists and has the correct access permission.
	SAW-A20103	File parse failed	Please check your input file has a valid column number.
merge	SAW-A30001	Parameter missing	Please check your input parameters. Some required parameters might be missed.
	SAW-A30002	File open failed	Please check the input file exists and has the correct access permission.
	SAW-A30003	File parse failed	Please check your mask file is in the correct file format. Only support .h5/.bin mask file.
	SAW-A30004	Fail to create output file	Fail to create output file. Please check your writing permission of the output directory.
	SAW-A30005	Fail to open input file	Fail to open input TXT file. Please check the file that exists and have the correct access permission.
	SAW-A30006	Out of index	Record coordinates exceed the expected range. Please check the input file is the output of mapping.
	SAW-A30007	Allocate memory error	Please check whether the range of the coordinates is too large, or you have run out of RAM. You may need to kill some jobs that use the same RAM or upgrade your hardware to get more RAM resources for your job.
register & rapidRegister	SAW-A40001	Parameter missing	Please check your input parameters. Some required parameters might be missed.
	SAW-A40002	File open failed	Please check the input file that exists and has the correct access permission. Or, please check whether a stitched panoramic TIFF (.tif or .tiff) image exists in the TAR.GZ.
	SAW-A40003	File parse failed	Please check your input file is in the correct file format. The -v input gene expression matrix should be either a tsv, barcode_gene_exp.txt, .gem.gz, or *raw.gef.
	SAW-A40004	Invalid data or data exception	Error data. Please check the file content. This error may arise because the -v input file is empty, the CZI file in TAR.GZ is invalid, or the QCPassFlag in IPR is 0.
	SAW-A40005	Tissue segmentation error	Abnormal tissue segmentation reference score in image preprocessing.
	SAW-A40006	IPR field missing	"Stitch/BGIStitch/StitchedGlobalLoc", does not exist.
	SAW-A40007	Tiled image missing	Please check the uncompressed image folder has tiled images.
imageTools	SAW-A40401	Parameter missing	Please check your imageTools merge input parameters. Some required parameters might be missed.
	SAW-A40402	File open failed	Please check the imageTools merge input file that exists and has the correct access permission.
	SAW-A40405	Invalid input	imageTools merge inputs of less than two images or more than three images.
	SAW-A40406	File pairing failed	Please check the imageTools merge input TIFF sizes are the same. Since the merge function is used for evaluating segmentation results, the input images are supposed to be the same in size and position (tissue position in the whole image).
	SAW-A40501	Parameter missing	Please check your imageTools overlay input parameters. Some required parameters might be missed.
	SAW-A40502	File open failed	Please check the imageTools overlay input file that exists and has the correct access permission.
	SAW-A40504	Invalid data or data exception	Error data. Please check the file content. This error may arise because the -c input IPR file does not include Stitch/TransformTemplate or Register/MatrixTemplate information
	SAW-A40601	Parameter missing	Please check your imageTools img2rpi input parameters. Some required parameters might be missed.
	SAW-A40602	File open failed	Please check the imageTools img2rpi input file that exists and has the correct access permission.
	SAW-A40605	Invalid input	imageTools img2rpi input -i and -g have different length. These two inputs are supposed to be paired.
	SAW-A40701	Parameter missing	Please check your imageTools ipr2img input parameters. Some required parameters might be missed.
	SAW-A40702	File open failed	Please check the imageTools ipr2imginput file exists and has the correct access permission. Or, please check whether a stitched panoramic TIFF (.tif or .tiff) image exists in the TAR.GZ.
	SAW-A40703	File parse failed	Please check your imageTools ipr2img input file is in the imageQC/imageStudio output TAR.GZ format.
	SAW-A40704	Invalid data or data exception	Error data. Please check the imageTools ipr2img input IPR file content. This error may arise because the CZI file in TAR.GZ is invalid, or the image has not either automatically or manually registered with the expression matrix. The second circumstance can be confirmed from IPR by checking whether the StereoResepSwitch/register is TRUE (has not performed automatic registration) or the ManualState/register is FALSE (has not performed manual registration). The third possible reason is the StereoResepSwitch/tissueseg is TRUE, therefore cannot output cell segmentation result.
	SAW-A40706	File pairing failed	Please check the shape of registered tissue segmentation images stored in the imageTools ipr2img input IPR are the same. Or please contact FAS/FBS for help.
tissueCut	SAW-A50001	Parameter missing	Please check your input parameters. Some required parameters might be missed
	SAW-A50002	File open failed	Please check the file that exists and has the correct access permission.
	SAW-A50003	File parse failed	Please check your input file is in the correct file format.
	SAW-A50004	Fail to create output file	Fail to create output file. Please check your writing permission of the output directory.
	SAW-A50005	Fail to write TIFF	Fail to write a TIFF file.
	SAW-A50006	h5AttrWrite error	Please check the H5 file attributes.
	SAW-A50007	h5DatasetWrite error	Please check the H5 file dataset.
cellCut	SAW-A60001	Parameter missing	Please check your input parameters. Some required parameters might be missed
	SAW-A60002	File open failed	Please check the file that exists and has the correct access permission.
	SAW-A60003	File parse failed	The file does not contain correct information. Please check the file format.
	SAW-A60110	Program version error	Please check your output GEF version. Your input GEF version might be too old.
	SAW-A60111	Call process error	e.g. "Please call freeRestriction first, or call restrictRegion function before restrictGene." This error arose because the invocation flow order was messed up. Please modify your invocation flow as prompted.
	SAW-A60120	Invalid data or data exception	Error data. Please check the file content.
	SAW-A60121	File information missing	Failed to read the file. Please check whether the file is damaged.
	SAW-A60122	File pairing failed	Please check the TIFF mask size is consistent with the size of expression matrix. Since the mask has been registered with the expression matrix, their sizes are supposed to be the same.
	SAW-A60130	Fail to create output file	Fail to create output H5 file. Please check your writing permission of the output directory or contact FAS/FBS for help.
	SAW-A60140	Allocate memory error	This error occurs when you run out of RAM. You may need to kill some jobs that use the same RAM or upgrade your hardware to get more RAM resources for your job.
	SAW-A60150	Dimensions of gene expression matrix did not match	Please contact FAS/FBS for help.
spatialCluster	SAW-A70001	Parameter missing	e.g. "-i or --gef_file is missing" Please check your input parameters. Some required parameters might be missed.
	SAW-A70002	File open failed	e.g. “cannot access /path/to/file: No such file or directory.” Please check the file that exists and has the correct access permission.
	SAW-A70005	Value error	e.g. "The bin size is out of range, please check the range of gef binsize is in [1,10,20,50,100,200,500]." Please reset the bin size as prompted. e.g. "Gene number less than 3000, please check your gef file" Please check the content of your GEF file, and make sure there are at least 3000 genes for clustering.
cellCluster	SAW-A70101	Parameter missing	e.g. "-i or --gef_file is missing" Please check your input parameters. Some required parameters might be missed.
	SAW-A70102	File open failed	e.g. “cannot access /path/to/file: No such file or directory.” Please check the file that exists and has the correct access permission.
	SAW-A70105	Value error	e.g. "The bin size is out of range, please check the range of gef binsize is in [1,10,20,50,100,200,500]." Please reset the bin size as prompted. e.g. "Gene number less than 3000, please check your gef file" Please check the content of your GEF file, and make sure there are at least 3000 genes for clustering.
saturation	SAW-A80001	Parameter missing	e.g. "-i is missing." Please check your input parameters. Some required parameters might be missed.
	SAW-A80002	File open failed	Please check the file that exists and has the correct access permission.
	SAW-A80003	File parse failed	Invalid GEF file. Please check the file format.
	SAW-A80004	Invalid data or data exception	e.g. "no data left after filter by coordinates." Please check the file content.
	SAW-A80005	Invalid data or data exception	e.g. "total map reads is 0, please check file format from --bcstat" Please check the file content of the input file as prompted.
	SAW-A80006	File pairing failed	e.g. "map reads less than annotated reads." Please check the input mapping statistical report and the count statistical report are from the same analysis.
	SAW-A80007	Plot error	Please contact FAS/FBS for help. PATH environment may not have python3.
	SAW-A80008	Fail to create output file	Fail to generate saturation file, please contact FAS/FBS for help.
report	SAW-A90001	Parameter missing	e.g. "-m or --barcodeMapStat is missing." Please check your input parameter as prompted. Some required parameters might be missed.
	SAW-A90002	File open failed	e.g. "cannot access *: No such file or directory." Please check the file that exists and has the correct access permission.
	SAW-A90003	File parse failed	JSON file format error. This error may arise because the input statistics files were not generated in the same SAW version as report. Or, the input mapping file prefix can not be parsed. Please contact FAS/FBS for help.
	SAW-A90004	Invalid data or data exception	e.g."information loss: fail to find 'bin_[size]' or 'ssDNA' in '*.rpi'." Please check the file content.
	SAW-A90005	Fail to create output file	Fail to create output file. Please check your writing permission of the output directory.
manualRegister	SAW-A40801	Parameter missing	Please check your input parameters. Some required parameters might be missed
	SAW-A40802	File open failed	Please check the file that exists and has the correct access permission. Or, please check whether the pre-registered image fov_stitched_transformed.tif exists in the input directory.
	SAW-A40803	File parse failed	Please check your input file is in the correct file format. The -v input gene expression matrix should be either a tsv, barcode_gene_exp.txt, .gem.gz, or *raw.gef.
	SAW-A40804	Invalid data or data exception	Error data. Please check the file content. This error may arise because the -v input file is empty. The second possible reason is that the gene expression matrix information in the IPR Register module (MatrixShape, Xstart, Ystart) does not match with the input GEF file (minX, minY, maxX, maxY), because the manual registration has to be processed on the identical matrix.

References

BGIResearch/SAW. Accessed October 13, 2021. https://github.com/STOmics/SAW

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