10x Genomics data are stored in three FASTQ files, besides the standard R1 and R2 reads, there is also a I1 file with some metadata. Sometimes however they are shipped in a single bam/cram file (e.g. the data from the Darwin Tree of Life). How can I convert the BAM or CRAM files to FASTQ?

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Apparently, the I3 file is not 10x specific thing. It is an index generated when demultiplexing any Illumina data. See this question for a nice explanation.

  • $\begingroup$ Isn't I1 the index read not unique to 10x data? But normally demultiplexing Illumina data with bcl2fastq does not generate these reads with default arguments $\endgroup$ Jun 8, 2021 at 20:12
  • $\begingroup$ @Chris_Rands (after a bit of googling) you are right. I just not aware of its existence before I run into this question on the tDToL GitHub. And then I thought I might as well put it here so it's easier to google it. $\endgroup$ Jun 8, 2021 at 22:44

2 Answers 2


All can be done with samtools. This is how the Darwin Tree of Life folks convert it:

samtools fastq -@4 -i \
  -1 ${sample}_S${tag}_L%03s_R1_001.fastq.gz \
  -2 ${sample}_S${tag}_L%03s_R2_001.fastq.gz \
  --i1 ${sample}_S${tag}_L%03s_I1_001.fastq.gz \
  --index-format i8 ${lane}.cram
  • $\begingroup$ I don't know if that solution will work with 10xGenomics files. You have to recreate R2 out of the contents of tags; there will be no reads with the read2 binary flag set. $\endgroup$
    – swbarnes2
    Jun 9, 2021 at 19:41
  • $\begingroup$ @swbarnes2 I just have seen this thread: github.com/darwintreeoflife/darwintreeoflife.data/issues/… and tried to make it more googlable (because I do think there will be quite a few people who will be downloading the tDToL data). $\endgroup$ Jun 10, 2021 at 9:39
  • $\begingroup$ I saw that thread too. I don't think it's right. samtools fastq -2 will work fine for bams from many applications, but not 10XGenomics. $\endgroup$
    – swbarnes2
    Jun 10, 2021 at 16:45
  • $\begingroup$ @kamil, can you post a couple of lines of the BAM file showing how tags for R1 and R2 appear in 10x data? I can probably quite easily add support for this in genozip.com/sam2fq.html $\endgroup$
    – Divon
    Jul 25, 2021 at 1:21

You'll want to use the 10x provided bamtofastq tool to preserve the indices properly: https://github.com/10XGenomics/bamtofastq


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