Hello everyone
I have 10X spatial transcriptome data from four samples (2 treated and 2 untreated). For 2 samples, transcriptome mapping rate is very less (< 30 %). Next I wanted to see sequence statistics of unmapped reads from bam file generated by spaceranger . I followed these steps :
1) extraction of unmapped reads
samtools view -f 4 possorted_genome.bam > possorted_genome_bam_unmapped.bam (file size 4 gb).
2) conversion of bam to fastq
bedtools bamtofastq -i possorted_genome_bam_unmapped.bam -fq unmapped_R1.fq -fq2 unmapped_R2.fq (0 bytes).
Last step gave me empty .fq files.
I would appreciate all the suggestions.
•
written
by
Omics data mining
https://bedtools.readthedocs.io/en/latest/content/tools/bamtofastq.html
-fq2 Creating two FASTQ files for paired-end sequences. When using this option, it is required that the BAM file is sorted/grouped by the read name. This keeps the resulting records in the two output FASTQ files in the same order. One can sort the BAM file by query name with samtools sort -n -o aln.qsort.bam aln.bam.
I tried to process my bam files as follows :
samtools sort -n -o possorted_genome_bam.qsort.unmapped.bam possorted_genome_bam_unmapped.bam
bedtools bamtofastq -i possorted_genome_bam_unmapped.bam -fq unmapped_R1.fq -fq2 unmapped_R2.fq
But still its giving me empty .fq files.
what is the output of
samtools view possorted_genome_bam_unmapped.bam | head
Here is the output :
A00556:201:HLTVVDRXY:2:1101:1063:2268 4 * 0 0 * *0 0 AAGCAGTGGTATCAAACGCAGAGTACATGGGTGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAGAAAAAGAATAAAAAAAAAAAATAA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFF,FFFFFFFFFFFFFFFF:,FFFF,FFF,,F:F,:F:,,F::::,:,F:FF,:F,,F:,::F,F,:::,,:FF:FF,,,F,F,,,,:F,:,:,,:,,:,,,,NH:i:0 HI:i:0 AS:i:86 nM:i:1 uT:A:1 xf:i:0 ts:i:28 li:i:0 BC:Z:TGCGCGGTTTQT:Z:FFFFFFFFFF CR:Z:CGCGCATGTTTGATTG CY:Z:FFFFFFFFFFFFFFFF CB:Z:CGCGCATGTTTGATTG-1 UR:Z:CTACTCTTGTAC UY:Z:FFFFFFFFFFFF UB:Z:CTACTCTTGTAC RG:Z:S3_C1_Complete:0:1:HLTVVDRXY:2
A00556:201:HLTVVDRXY:2:1101:1063:3051 4 * 0 0 * *0 0 AAGCAGTGGTATCAACGCAGAGTACATGGAAAAAAAATTCTTTTAATGTGGAAACAATAAATTTCACAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,:FFFFFFFF:F:FFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFF:F:F::FF:FFFFFFFFFFF::FFFFFFFFF,::FFF:F:F:F,F:F:F:FF::F::F::,:FF,FF,NH:i:0 HI:i:0 AS:i:82 nM:i:2 uT:A:1 xf:i:0 ts:i:27 li:i:0 BC:Z:TGCGCGGTTTQT:Z:FFFFFFFFFF CR:Z:TTGGATATCGTCTACG CY:Z:FFFFFFFFFFFFFFFF CB:Z:TTGGATATCGTCTACG-1 UR:Z:GCTTGTGTTGTC UY:Z:FFFFFFFFFFFF UB:Z:GCTTGTGTTGTC RG:Z:S3_C1_Complete:0:1:HLTVVDRXY:2
A00556:201:HLTVVDRXY:2:1101:1063:3552 4 * 0 0 * *0 0 AAGCAGTGGTATCAACGCAGAGTAATGGGGAGTGCGGGTAGGAGCCGTGAGGTGCTTCTCTGCTGTGACAAACGACCCTGTCTGTCCGTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA FF::FF,FF:FFFFFF::FFF:,FFFFFFFF::FFFFF:,:FFFFFF:FF,,::F,FFFF:FFFFF:FFFFFFFFFFFFF,FFFFF:FFFF:::FFFFFFFFFFFFFFFFF,F,FFFF:FFFFFFF:F::F,,,:F,,,,F,,F,:,,FF,NH:i:0 HI:i:0 AS:i:62 nM:i:0 uT:A:1 xf:i:0 ts:i:27 li:i:0 BC:Z:TGCGCGGTTTQT:Z:FFFF,:FFF, CR:Z:AGAAGTGATTCGTGAT CY:Z:,FFFFFFFFFFFFFFF CB:Z:AGAAGTGATTCGTGAT-1 UR:Z:ACCGGAAGTATA UY:Z:FF:FFFFFFFFF UB:Z:ACCGGAAGTATA RG:Z:S3_C1_Complete:0:1:HLTVVDRXY:2
A00556:201:HLTVVDRXY:2:1101:1063:3709 4 * 0 0 * *0 0 AAGCAGTGGTATCAACGCAGAGTACATGGGCCCTGGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA FFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,F,FFFFF::FFFFF,FF,FFF:FFFFF,FFFF,FF::FFF,F,:,F:F,:FF,:,:FF,F,NH:i:0 HI:i:0 AS:i:102 nM:i:0 uT:A:3 xf:i:0 ts:i:30 li:i:0 BC:Z:TGCGCGGTTT QT:Z:FFFFFFFFFF CR:Z:GTTGAACCGGTTCCAT CY:Z:FFFFFFFFFFFFFFFF CB:Z:GTTGAACCGGTTCCAT-1 UR:Z:TACGGGATAGTT UY:Z:F:FFFF:FFFFF UB:Z:TACGGGATAGTT RG:Z:S3_C1_Complete:0:1:HLTVVDRXY:2
A00556:201:HLTVVDRXY:2:1101:1063:4617 4 * 0 0 * *0 0 AAGCAGTGGTATCAACGCAGAGTACATGGGAAAATAAAATCCTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFFFFFFFFFFFFFFF:FF:FFFFFFFFFFFF:FFFF:,F:FF:F,FFFFFFFF:FF:FFFFFFF:F:,:F,:F,FF:FF:F:F,:F::F:,F:FF,F,:F,::FF:,NH:i:0 HI:i:0 AS:i:97 nM:i:6 uT:A:1 xf:i:0 ts:i:30 li:i:0 BC:Z:TGCGCGGTTTQT:Z:FFFFFFFFFF CR:Z:CGATCCTCGCAACATA CY:Z:FFFFFFFFFFFFFFFF CB:Z:CGATCCTCGCAACATA-1 UR:Z:TGGTTTGGGCAC UY:Z:FFFFFFFFFFFF UB:Z:TGGTTTGGGCAC RG:Z:S3_C1_Complete:0:1:HLTVVDRXY:2
A00556:201:HLTVVDRXY:2:1101:1063:5869 4 * 0 0 * *0 0 GTGGTATCAACGCAGAGTACATGGGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAGAAAAAAAAAAAAAAAG FFFFFFFFFFFF,FFFFF:FFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFF,F,:FF:FFFFF:F:FFFF:,FFFFFF,F:FFF,F,:FFFFFF:F,F:,,,FF:,,,,,,FF:,,,FFF:,F:,,,F,:,,,:::,,,,,,:F:,,,,NH:i:0 HI:i:0 AS:i:88 nM:i:0 uT:A:1 xf:i:0 ts:i:25 li:i:0 BC:Z:TGCGCGGTTTQT:Z:FFFFFFFF,F CR:Z:CTCCTGTTCAAGGCAG CY:Z::FFFFFFFFFFFFFF: CB:Z:CTCCTGTTCAAGGCAG-1 UR:Z:CTCCCTCGCGCA UY:Z::F,FFFFFFFFF UB:Z:CTCCCTCGCGCA RG:Z:S3_C1_Complete:0:1:HLTVVDRXY:2
A00556:201:HLTVVDRXY:2:1101:1063:5932 4 * 0 0 * *0 0 AAGCAGTGGTATCAACGCAGAGTACATGGGAAAAGTGAATCTTTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFF,F,FFFFFF,F::FFFFFFFFFFFFF:FFFF::FFFF:FF:,,FFFF:FFFFFFFFFF,F,,,F,:,F::,:,::,F:,,FFF,,,FF,,:F:FFF,:FF::F:,F::,NH:i:0 HI:i:0 AS:i:66 nM:i:0 uT:A:1 xf:i:0 ts:i:30 li:i:0 BC:Z:TGCGCGGTTTQT:Z:FFFFFFFFFF CR:Z:CTATTCATGTGTCCCA CY:Z:FFF:FF:FFFFFFFFF CB:Z:CTATTCATGTGTCCCA-1 UR:Z:TTGTTGTTGAAT UY:Z::FF:F::F,,FF UB:Z:TTGTTGTTGAAT RG:Z:S3_C1_Complete:0:1:HLTVVDRXY:2
all those reads are unmapped flag=4 , but anyway, those reads are SINGLE-END reads ( flag 1 = read paired is unchecked). So using -fq -fq2 is meaningless
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