empty fragment file

[rojinsafavi]

Hi Tim,
So I created a new column for barcode info (using sam), and then convert the sam to the bam file and indexed it. I still get no fragment file.
The head:

7001113:989:HTKVHBCX2:2:1105:15600:5528:77:15:82:15:CGGTATTTGG	0	chr1	3000049	1	23M	*	0	0	TCTTTGAAGGTCTGGTAGAACTC	DDDDDIIIIIIIIIIIIIIIIII	AS:i:0	CB:Z:77158215
7001113:991:HVWNKBCX2:1:2110:2683:18182:87:93:72:36:CAGGTATGGC	0	chr1	3000132	39	53M	*	0	0	GACTATTGATGACTGCCTCTATTTCTTTAGGGGAAATGGGACTTTTAGTCCADDDDCHIIHGFHIIIIIIIIIIHHHEHIIIIIIIDDGHHHDHHIIIIIIIIHI	AS:i:0	CB:Z:87937236
7001113:990:HTKL3BCX2:2:2111:14944:4116:87:93:72:36:CAGGTATGGC	0	chr1	3000134	38	52M	*	0	0	CTATTGATGACTGCCTCTATTTCTTTAGGGGAAATGGGACTTTTAGTCCATGDDDDDIIIIIIIIIIIIIIIIIIIGIIIIIIIDFHHHHHIIIIIIIIIHIII	AS:i:0	CB:Z:87937236
7001113:991:HVWNKBCX2:2:2105:19568:57493:53:23:77:35:CACTATTTTG	0	chr1	3000159	0	52M	*	0	0	GGGGGGGCATGGGACTTTTAGTCCATGAATCTGATCCTGATTTAGCTTTGGTDDDDDIIIIIIIIIIIIIHIIIIIIIIIIIIIIIIIIIIHEHIIHGIIIIIH	AS:i:-22	CB:Z:53237735
7001113:993:HVWMKBCX2:2:1211:1360:3780:53:23:77:35:CACTATTTTG	0	chr1	3000353	37	53M	*	0	0	GTTAATTATAGTACAGTCCCTATGCCCTCTAGTTAGTCTGGCTAAGGGTTTADDDDDIIHIIIIIIIIIIIHIIIIIIIIIIIIHIGIHIIHHIIIHHIIIHIII	AS:i:0	CB:Z:53237735
7001113:991:HVWNKBCX2:2:2211:8406:75832:21:06:91:12:TCATCTTTGT	16	chr1	3000464	1	52M	*	0	0	TCTTTTTGTTTCCACTTGGTTGATTTCAGCTCTGAGTTTGATTATTTCCTGCIHIHIIIIHHFF@EHIHGCHF<1IHFEEHEIHIIHIIHHHFIIGIHIDDDDD	AS:i:0	CB:Z:21069112
7001113:989:HTKVHBCX2:2:1211:2292:73091:90:80:26:12:CTGTACGGCT	0	chr1	3000559	42	53M	*	0	0	CTTCTAGATTTGCTGTCAGGCTGCTAGTGTATACTCTAGTTTCCTTTTGGAGDDCDDIIIIIIIIHIIIIIIIIIIIIIHIIIIIIIIHIIIIIIIIIIIIHIII	AS:i:0	CB:Z:90802612
7001113:991:HVWNKBCX2:1:2205:17457:98814:90:80:26:12:CTGTACGGCT	0	chr1	3000633	30	53M	*	0	0	CTCTTAGGACTGCCTCATTGTGCCCCATATGTTTGGCTATGTTGTGGATTTADDDDDIIIIIIIIIIIIIIIIIIIIIIIHIIIIIIIIIIIIIIIIIIIIIIII	AS:i:0	CB:Z:90802612
7001113:989:HTKVHBCX2:1:2216:4512:6199:90:80:26:12:CTGTACGGCT	0	chr1	3000747	32	52M	*	0	0	ATTAAGTAGAGTATTGTTCAGTTTCCAGGTGAATGTTGGCTTTCTATTATTTDDDDDIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGIIIIIH	AS:i:0	CB:Z:90802612
7001113:989:HTKVHBCX2:2:2209:10226:10613:58:87:87:11:TCGAATTTGT	0	chr1	3000919	32	51M	*	0	0	ATTTGGTACTGAGAAGAAGGTATATATCCTTTTGTCTTATGATAAAATGTT	DDDDDIIIIIIIHIIIIIHIIIHIIIIIIIIIIIIIIGIIIIIIIIIIIII	AS:i:0	CB:Z:58878711

the sinto code:
sinto fragments -b merge_CB.bam -f fragment

[timoast]

Can you share a link to a downsampled version of the bam file that reproduces the issue? You can email a link to tstuart@nygenome.org

[rojinsafavi]

I just did, thank you very much

[timoast]

It looks like the BAM file is single-end, each read name only appears once in the file. A paired-end BAM is required for this function.

[rojinsafavi]

@timoast I also have access to the fast file (and it is actually a paired-end read fastqs). Do you suggest going from fastqs to bam files then? I have 3 replicates, which means I will end up with 3 bam files. Do you suggest to merge them together at the end and make a fragment file out of it?

[timoast]

Sure, you could map the fastqs to create a new bam file. Not sure why the bam is single-end if you had a paired fastq file. There's an example here of how to go from fastq to fragment file: https://timoast.github.io/sinto/scatac.html