# Questions regarding Nanopore sequencing analysis

I am new to Nanopore sequencing analysis. I have a couple of questions regarding it which are as follows:

1. How do I know if my fast5 file is multiread or single read file?
2. Is there a way to combine all the fast5 files into a single fast5 file?
3. How can I extract the flowcell type and sequencing kit information from the fast5 file? Is there a command that I can use to extract it? I know I can use the following command to retrieve it:
h5dump -a /UniqueGlobalKey/context_tags/sequencing_kit -a /UniqueGlobalKey/context_tags/flowcell_type <fast5 file>


However, it works well for single read file and not for multiread files. Please correct me if I am wrong. Thanks!

1. The quickest way to know if it's a single or multi fast5 is the file name. If it mentions channels and read numbers (e.g. ..._read_9_ch_471_..., then it's a single read file. Alternatively, the multi-read files have only read IDs at the top level (i.e. read_<32-character-string-with-additional-dashes>). Example:

$h5ls <read_file_name>.fast5 | head read_0115573a-042a-42a3-b4e2-f0338b4a6c66 Group read_015b870d-8a03-4f22-a73e-fb9f833b94c7 Group read_0167393a-dede-4a27-91af-494622b63261 Group read_01e658de-853b-4f58-93ad-87fbd735254c Group read_0202acc2-33f4-4d29-9dac-c7f80c758c86 Group read_022d68f0-6f8e-4124-bfb0-11262efa4a67 Group read_0258a3c9-4ffe-42ab-a42d-89466fcefbf5 Group read_032da6db-e84a-43ec-8c25-bcaead992066 Group read_03383754-be43-420b-87fc-60c2eb833963 Group read_036b4824-bb0a-42dd-ade0-29d89112ff07 Group  2. ONT provide a single_to_multi program for converting to the multi-read format. Details can be found on this github page. Here's a quick example of how it could be run: $ single_to_multi_fast5 --input_path fast5 --save_path fast5_multi \
--filename_base multi_file_myRunID --batch_size 4000 --recursive


3a. For multi-fast5 files, the kit/cell attributes are in the context_tags group of the file (because a multi-fast5 file could contain fast5 elements from multiple different sequencing runs with different run parameters):

$h5dump -g /read_<readID>/context_tags multi_file_myRunID_0.fast5 | \ grep -A 11 -e 'sequencing_kit' -e 'flowcell_type' ATTRIBUTE "flowcell_type" { DATATYPE H5T_STRING { STRSIZE 11; STRPAD H5T_STR_NULLTERM; CSET H5T_CSET_ASCII; CTYPE H5T_C_S1; } DATASPACE SCALAR DATA { (0): "flo-min106" } } -- ATTRIBUTE "sequencing_kit" { DATATYPE H5T_STRING { STRSIZE 11; STRPAD H5T_STR_NULLTERM; CSET H5T_CSET_ASCII; CTYPE H5T_C_S1; } DATASPACE SCALAR DATA { (0): "sqk-pbk004" } }  3b. For single fast5 files (at least, in the most recent version of them), they are found in the UniqueGlobalKey group: $ h5dump -g /UniqueGlobalKey single_file_read_X_ch_Y_strand.fast5 | \
grep -A 11 -e 'sequencing_kit' -e 'flowcell_type'
ATTRIBUTE "flowcell_type" {
DATATYPE  H5T_STRING {
STRSIZE 11;
CSET H5T_CSET_ASCII;
CTYPE H5T_C_S1;
}
DATASPACE  SCALAR
DATA {
(0): "flo-min106"
}
}
--
ATTRIBUTE "sequencing_kit" {
DATATYPE  H5T_STRING {
STRSIZE 11;
CSET H5T_CSET_ASCII;
CTYPE H5T_C_S1;
}
DATASPACE  SCALAR
DATA {
(0): "sqk-pcs108"
}
}


[note that this file structure may change in the future]

• I have another question. What is an exact difference between multiread or single read file? Where can I get its format? Feb 19 '19 at 5:35
• I’d start on ONT’s website & check out the file format documentation. I think their announcement is a good place to begin.
– Scot
Mar 3 '19 at 19:18