FASTA & FASTQ

There seems to be a never ending source of file formats out there. Documenting past obsolete formats, one would assume a point at which there are no more to find, but in reality more are re-discovered everyday by the Digital Preservation community. When it comes to more modern formats, it seems more are invented everyday, too many to keep up with identification. Document one, 10 more pop up, it seems never-ending. Such is the case for scientific formats, including sequencing formats.

I was speaking with a colleague from another institution the other day and a file format was mentioned I hadn’t heard of before. It seems many of their scientific data was stored in a format called FASTA “Fast A” (“fast-aye”). This format specifically stores DNA sequence data and is used quite a bit, it seems. I was even more surprised the next day when I went to process some new submissions for our repository only to find one submission contained three FASTA files. I love researching file formats, but sometimes in order to understand the format structure you have to know something about the content as well. Let’s explore the FASTA and FASTQ file formats. If you would like to take a peek at the Human Genome in FASTA, go here.

Both the FASTA and FASTQ formats are text based and have a simple structure. Identification of each of these should be pretty simple, but to avoid conflicts with other formats, the signature might have to be more complex.

The FASTA format is well documented as many in the scientific community use it. Basically the format starts with the greater than “>” character followed by a description, a new line character, then the sequence. For example:

>MCHU - Calmodulin - Human, rabbit, bovine, rat, and chicken
MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTID
FPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREA
DIDGDGQVNYEEFVQMMTAK*

Pretty straight forward, but so much of the format can be variable, a simple signature would clash with too many other formats. There are some rules with what characters can be used in the sequence so it might be possible to limit the signature to only allow certain characters. At first I thought it might only be able to contain the standard characters representative of adenine (A), cytosine (C), guanine (G), and¬†thymine¬†(T), but as it turns out the FASTA format can contain Nucleic Acid Code’s and Amino Acid Code’s. These codes allow more than the four I was expecting, but do limit what can be represented.

Take the NCBI Sequence Viewer for a spin and download some data as FASTA.

The FASTQ format adds more structure and is more limiting, but also presents some challenges. Here is a sample of its structure:

@SEQ_ID
GATTTGGGGTTCAAAGCAGTATCGATCAAATAGTAAATCCATTTGTTCAACTCACAGTTT
+
!''*((((***+))%%%++)(%%%%).1***-+*''))**55CCF>>>>>>CCCCCCC65

Instead of a greater than symbol, the FASTQ format uses an “@” symbol followed by an identifier. The identifier can be basically anything and as long as needed. There is a newline character followed by the DNA sequence, which is only the four characters I have heard before. It can contain an A, C, G, T, or N. The “N” can represent an unidentified nucleotide or indicate that the software was unable to make a basecall. A newline character again and the “+” symbol. This is place before the fourth line with is a quality score and is the same number of characters as the sequence.

See what I mean when you have to learn about the context of a format in order to make a proper signature!

One of the problems I am left with is how to determine how many of the sequence characters to use in the signature to not have any conflicts. Too few and it might conflict with another format or simple text file. Too many and the signature gets complicated and may exclude a short sequence file. As far as I can tell there is no set minimum or maximum for the sequence. Not sure what the genome for Pinus Taeda would look like in FASTA with 22.18 billion base pairs. The other problem is often times these formats are compressed into a GZIP file, so they need to be extracted before identification.

These two formats are just a couple of the many sequencing formats being used in the bioinformatics community. I am sure others will pop up in the future. Until then, I have with the help of others put together a signature which seems to work well for the samples and data sets we have access to. Take a look at my GitHub for the signature proposal. If you find any issues, let me know!

AskSam

I was recently asked to look at a set of files with the extension of .ASK. A quick little search led me to find they belong to AskSam which was a free-form database software often used by researchers and libraries as early as 1985. The first few versions of Access Stored Knowledge via Symbolic Access Method were released for DOS and later Windows. The company askSam Systems disappeared around 2015.

The AskSam software competed with other personal information managers with unstructured data storage and retrieval. It was used to keep track of e-mail, special collections, letters, articles, web sites, etc. It could index all the contents and make searching and retrieval easy. By setting up fields the data could be exported to delimitated text. The software also appears to have been localized in German, but file format is the same.

AskSam had many import filters which included:

  • Microsoft Word
  • WordPerfect
  • Text (ASCII files)
  • HTML Files (from the Internet)
  • RTF Files (Rich Text Format)
  • Eudora E-Mail
  • Microsoft Outlook
  • Microsoft Outlook Express
  • Text delimited files – Comma Separated Values, Fixed position, etc.
  • dBASE
  • FoxPro
  • Paradox
  • Microsoft Access
  • Microsoft Excel

AskSam has its own proprietary format to store the database using the .ASK extension. They appear to have a 256 byte header. All the DOS versions of the software use the simple BOF string of “askSam”.

hexdump -C TEST.ASK       
00000000  61 73 6b 53 61 6d 00 00  00 00 00 07 0f 01 00 00  |askSam..........|
00000010  01 00 00 00 00 01 00 05  00 37 00 02 00 00 00 01  |.........7......|
00000020  33 00 32 00 00 00 00 00  50 00 00 00 00 00 00 00  |3.2.....P.......|
00000030  00 00 00 00 00 00 00 00  00 00 00 00 00 00 00 00  |................|
*
000000d0  00 14 00 01 00 00 01 00  00 00 00 00 00 00 00 00  |................|
000000e0  00 00 00 00 00 01 00 00  00 00 03 1d 42 00 01 00  |............B...|
000000f0  00 13 01 00 00 00 00 01  00 00 00 00 00 00 00 00  |................|
00000100  00 00 00 00 f6 00 00 00  00 54 65 73 74 01 01 01  |.........Test...|
00000110  01 01 00  

When the first Windows version came out in 1993, the header changed to the logical string:

hexdump -C DOS-WIN.ASK | head
00000000  61 73 6b 77 69 6e 00 00  00 00 00 07 0f 01 00 04  |askwin..........|
00000010  01 00 00 00 01 01 00 05  01 37 03 00 00 00 00 01  |.........7......|
00000020  64 00 32 2e 01 4e 00 00  a0 00 00 00 00 00 00 00  |d.2..N..........|
00000030  00 00 00 00 00 00 00 00  00 00 00 00 00 00 00 00  |................|
00000040  00 00 00 00 00 00 00 00  00 00 00 00 00 76 43 00  |.............vC.|
00000050  00 8c 00 00 00 00 00 00  00 00 00 00 00 01 00 00  |................|
00000060  00 01 00 00 00 00 00 00  00 00 00 00 00 00 00 00  |................|
00000070  00 41 72 69 61 6c 00 72  20 4e 65 77 00 00 00 00  |.Arial.r New....|
00000080  00 00 00 00 00 00 00 00  00 00 00 00 00 00 00 00  |................|
00000090  00 00 00 00 00 00 00 00  00 00 5b 3a 00 10 10 10  |..........[:....|

With Version 2 for Windows we start seeing a slightly different header:

hexdump -C AS2W-S01.ASK 
00000000  61 73 6b 57 69 53 00 00  00 00 00 07 0f 01 00 04  |askWiS..........|
00000010  01 00 00 00 01 01 00 05  00 37 03 00 00 00 00 01  |.........7......|
00000020  c8 00 32 2f 02 4c 00 00  a0 00 00 00 00 00 00 00  |..2/.L..........|
00000030  00 00 00 00 00 00 00 00  00 00 00 00 00 00 00 00  |................|
*
00000070  00 43 6f 75 72 69 65 72  20 4e 65 77 00 00 00 00  |.Courier New....|
00000080  00 00 00 00 00 00 00 00  00 00 00 00 00 00 00 00  |................|
00000090  00 00 00 00 00 00 00 00  00 00 5b 3a 00 10 10 14  |..........[:....|
000000a0  14 02 00 00 0a 00 00 00  00 00 00 00 00 00 00 00  |................|
000000b0  00 00 00 00 00 00 00 00  00 00 00 00 00 00 00 00  |................|
000000c0  00 00 00 00 00 00 00 00  00 00 00 60 00 00 00 00  |...........`....|
000000d0  05 00 00 00 00 00 01 00  00 00 00 00 00 00 00 00  |................|
000000e0  00 00 00 00 00 01 00 00  00 00 00 00 00 00 01 00  |................|
000000f0  00 1d 01 00 00 00 00 01  00 00 00 00 00 00 0a 00  |................|
00000100  00 00 00 00 f6 00 00 00  0a 54 65 73 74 69 6e 67  |.........Testing|
00000110  20 20 00 0a 01 09 10 c0  14 14 42 07 01           |  ........B..|

Then all samples from version 4 to the final version 7 all have the same header, although I know there is some features in the later versions that make them incompatible, there isn’t a easy way to identify the different versions after version 4.

hexdump -C Asksam4-s01.ask | head
00000000  61 73 6b 77 34 30 00 00  00 00 25 00 00 00 00 00  |askw40....%.....|
00000010  00 00 00 00 00 00 00 00  00 00 00 00 00 00 00 00  |................|
*
000000f0  00 00 00 00 00 00 00 00  00 00 02 00 00 00 e5 38  |...............8|
00000100  0c 3a 67 31 4d 38 dd b5  9c 65 00 00 00 00 90 01  |.:g1M8...e......|
00000110  00 00 01 01 0c 43 00 00  00 00 00 00 be 00 00 00  |.....C..........|
00000120  24 14 00 00 00 00 00 00  10 14 00 00 00 00 00 00  |$...............|
00000130  00 00 00 00 00 00 00 00  00 00 00 00 00 00 00 00  |................|
*
00000150  7b 4c 00 00 00 00 00 00  af 4f 00 00 00 00 00 00  |{L.......O......|

hexdump -C AskSam6-s01.ask | head
00000000  61 73 6b 77 34 30 00 00  00 00 38 00 00 00 00 00  |askw40....8.....|
00000010  00 00 00 00 00 00 00 00  00 00 00 00 00 00 00 00  |................|
*
000000f0  00 00 00 00 00 00 00 00  00 00 02 00 00 00 21 f1  |..............!.|
00000100  ad 41 61 9f c0 39 cd 4a  af 65 00 00 00 00 58 02  |.Aa..9.J.e....X.|
00000110  00 00 01 01 84 2e 00 00  00 00 00 00 be 00 00 00  |................|
00000120  24 14 00 00 00 00 00 00  50 13 00 00 00 00 00 00  |$.......P.......|
00000130  00 00 00 00 00 00 00 00  00 00 00 00 00 00 00 00  |................|
00000140  00 00 00 00 00 00 00 00  00 00 00 00 c6 5b 00 00  |.............[..|
00000150  ba 33 00 00 00 00 00 00  53 33 00 00 00 00 00 00  |.3......S3......|

hexdump -C AskSam7-s01.ask | head
00000000  61 73 6b 77 34 30 00 00  00 00 87 04 00 00 00 00  |askw40..........|
00000010  00 00 00 00 00 00 00 00  00 00 00 00 00 00 00 00  |................|
*
000000f0  00 00 00 00 00 00 00 00  00 00 02 00 00 00 b2 fd  |................|
00000100  b5 47 61 9f c0 39 5c 4b  af 65 00 00 00 00 bc 02  |.Ga..9\K.e......|
00000110  00 00 01 01 db 34 00 00  00 00 00 00 be 00 00 00  |.....4..........|
00000120  24 14 00 00 00 00 00 00  50 13 00 00 00 00 00 00  |$.......P.......|
00000130  00 00 00 00 00 00 00 00  00 00 00 00 00 00 00 00  |................|
*
00000150  aa 39 00 00 00 00 00 00  de 3c 00 00 00 00 00 00  |.9.......<......|

Even though everything after version 4 for Windows has the same header, files create din version 7 will not open in version 6. There must be some additional byte sequences which identify the files with the version which created the file. I have been unable to located the free askSam 7 viewer, but here is a link to the version 6 free viewer. It runs in the latest Windows OS. If you open an older version it will ask you to upgrade your file, so be sure to keep a copy of your original.

Once you have your ASK Database opened, you can export to a few formats, an RTF or a delimitated text file based on fields you have entered in the form. Word of warning, if you entered a password to protect modifying of your data in an earlier version, you have to re-enter the password in order to open/upgrade the file, but the viewer will not open password protected files, you will need the full version.

Here are two files created in AskSam 5.11 DOS, one without a password one with. You can see the 16 byte hex values from offset 41 to 57 are zeros in the file with no password and full of values in the protected file. I’m sure someone with more skills could figure out the encryption.

hexdump -C AS5-OPEN.ASK 
00000000  61 73 6b 53 61 6d 00 00  00 00 00 07 0f 01 00 00  |askSam..........|
00000010  01 00 00 00 00 01 00 05  00 37 00 02 00 00 00 01  |.........7......|
00000020  33 00 32 00 00 00 00 00  50 00 00 00 00 00 00 00  |3.2.....P.......|
00000030  00 00 00 00 00 00 00 00  00 00 00 00 00 00 00 00  |................|
*
000000d0  00 14 00 01 00 00 01 00  00 00 00 00 00 00 00 00  |................|
000000e0  00 00 00 00 00 01 00 00  00 00 03 1d 42 00 01 00  |............B...|
000000f0  00 13 01 00 00 00 00 01  00 00 00 00 00 00 00 00  |................|
00000100  00 00 00 00 f6 00 00 00  00 54 65 73 74 69 6e 67  |.........Testing|
00000110  01 01 00                                          |...|

hexdump -C AS5-PASS.ASK 
00000000  61 73 6b 53 61 6d 00 00  01 00 00 07 0f 01 00 00  |askSam..........|
00000010  01 00 00 00 00 01 00 05  00 37 00 02 00 00 00 01  |.........7......|
00000020  33 00 32 00 00 00 00 00  50 66 5f 14 66 42 53 40  |3.2.....Pf_.fBS@|
00000030  42 71 29 59 6a 61 62 60  6e 00 00 00 00 00 00 00  |Bq)Yjab`n.......|
*
000000d0  00 14 00 01 00 00 01 00  00 00 00 00 00 00 00 00  |................|
000000e0  00 00 00 00 00 01 00 00  00 00 03 1d 42 00 01 00  |............B...|
000000f0  00 13 01 00 00 00 00 01  00 00 00 00 00 00 00 00  |................|
00000100  00 00 00 00 f6 00 00 00  00 54 65 73 74 69 6e 67  |.........Testing|
00000110  01 01 00                                          |...|

You can check out my samples and my recommendation to PRONOM on my Github page.