Summary: In a sampling of 20 reads that yet another chimeric read detector (YACRD) detected in a run of E. coli (run X0161), 20 are true positives. The same results are seen on a metagenome run of Actino Mock (run X0163). No false positives are seen. The rate of true and false negatives is unknown. The github repository for YACRD can be viewed here: https://github.com/natir/yacrd. The repository for dictionaryChimeric.py is here: https://github.com/JGI-Bioinformatics/yacrd_analysis.

Explanation of code: The output of dictionaryChimeric.py is a new fastq file with all chimeric reads split out into separate reads. This new fastq file can now be used for analysis. The program takes the output of running YACRD sent to a txt file. This YACRD text file is parsed and stored in a dictionary where the read name is the key and the value is the rest of the output from YACRD for that read. For each line in the fastq file that is to be used, a check is done to see if that line what flagged by YACRD as being chimeric or "not covered". If it is anything other than chimeric, nothing is done (i.e. no reads are written to the new fastq file that is being generated). If it is chimeric, then the read is split up into individual reads where there is sufficient coverage, and these reads are written to the new fastq file. If the line in the original fastq file was not flagged by YACRD, then the whole read is written to the new fastq file as is, because it is a valid read and nothing needs to be done. Below this page is data from the E. coli run analysis of YACRD's impact on true/false positives, as well as that of Actino Mock.

Running the program: In order to run dictionaryChimeric.py, you will first need to run minimap2 and then YACRD. The commands I used are as follows: Run minimap2: shifter --image=robegan21/minimap2 minimap2 -x ava-ont X0163_small.fastq X0163_small.fastq > smallMapping.paf X0163_small.fastq is the original fastq smallMapping.paf is the mapping file I want to create Run yacrd: yacrd -f smallMapping.paf -i smallMapping.paf -o output.fastq > smallYacrd.txt The paf file must be the paf file you created when running minimap2 Run dictionaryChimeric.py on NERSC: source activate yacrd conda install biopython python dictionaryChimeric.py > smallOutput.txt smallOutput.txt is a file that contains useful printed information, including each read's name, the number of pieces that the read will be separated into, the YACRD data for each piece (i.e. length, starting index, and ending index of each region), and the name of each new read The file smallOutput.fastq is the resulting fastq file that can now be used for analysis

Results from E. coli run (X0161): on newSmallFastq: 380,000 reads 4296 are chimeric TP = true positive FP = false positive

Chimeric read: 50a2b172-651f-4333-ae6d-7f2bda586a80_Basecall_1D_template 3400713 152669 ref distance: 3248044 read distance: 6253-6249 = 4 difference: 3248040 2 pieces conclusion: TP Chimeric read: e0206847-2acb-4d7b-9844-26d218d6aa9b_Basecall_1D_template 1111429 429948 ref distance: 681481 read distance: 385-378 = 7 difference: 681474 2 pieces conclusion: TP Chimeric read: 2e95da4b-9d51-4eaa-a1cb-815ae3724469_Basecall_1D_template 3888829 3889169 ref distance: 340 read distance: 700-694 = 6 difference: 334 2 pieces conclusion: TP Chimeric read: 4c3f327f-5ff8-4771-a9e6-9dfd4bfc2033_Basecall_1D_template 3243801 4068657 ref distance: 824856 read distance: 435-417 = 18 difference: 824838 2 pieces conclusion: TP Chimeric read: d8ec6051-ab6b-4cf6-9a6e-d4fb8dd1b05e_Basecall_1D_template 3400711 2407703 ref distance: 993008 read distance: 21577-20756 = 821 difference: 992187 2 pieces conclusion: TP Chimeric read: bb09585b-55b8-44ad-b929-e0e22e776035_Basecall_1D_template 504434 3312064 ref distance: 2807630 read distance: 514-385 = 129 difference: 2807501 2 pieces conclusion: TP Chimeric read: f6a24bd9-19a7-497c-8542-8834a903a9c2_Basecall_1D_template 1077570 1078140 ref distance: 570 read distance: 882-873 = 9 difference: 561 2 pieces conclusion: FP? Chimeric read: 328c98ff-cb8e-40bd-837a-d987f41ebbcf_Basecall_1D_template 3954929 2044402 ref distance: 1910527 read distance: 635-627 = 8 difference: 1910519 2 pieces conclusion: TP Chimeric read: 14c0a8fe-ec0f-4e6f-bb6b-f5466fa8f2be_Basecall_1D_template 4096222 1031137 ref distance: 3065085 read distance: 1561-1545 = 16 difference: 3065069 2 pieces conclusion: TP Chimeric read: 5285c16c-c99c-451e-93ce-ecb87d82255d_Basecall_1D_template 4614982 770017 ref distance: 3844965 read distance: 3160-3138 = 22 difference: 3844943 2 pieces conclusion: TP Chimeric read: c8008c2d-f2fe-4656-a42b-5affcf10b5fe_Basecall_1D_template 2975146 2321423 ref distance: 653723 read distance: 3960-3947 = 13 difference: 653710 2 pieces conclusion: TP Chimeric read: 727fcf64-11e7-4b65-b5ee-4cd351c2a0d2_Basecall_1D_template 2703702 3967537 ref distance: 1263835 read distance: 785-784 = 1 difference: 1263834 2 pieces conclusion: TP Chimeric read: b6584aec-5d4e-4b84-a973-d80f6b280d43_Basecall_1D_template 1407235 779166 ref distance: 628069 read distance: 541-539 = 2 difference: 628067 2 pieces conclusion: TP Chimeric read: edc14709-0652-4317-b017-0cfd6fab2494_Basecall_1D_template 3799432 3799667 ref distance: 235 read distance: 18610-18601 = 9 difference: 226 2 pieces conclusion: FP? Chimeric read: 8414a449-2caa-4ece-a06d-632e1306c326_Basecall_1D_template 2391354 1614167 ref distance: 777187 read distance: 66316-66277 = 39 difference: 777148 2 pieces conclusion: TP Chimeric read: 2b4fea49-75e0-4911-bcdb-2cb7507fa1b5_Basecall_1D_template 177889 178525 ref distance: 636 read distance: 3766-3761 = 5 difference: 631 2 pieces conclusion: TP Chimeric read: bf10b8f5-276f-4912-b33c-b9d79096a8dd_Basecall_1D_template 842171 833019 ref distance: 9152 read distance: 11589-11233 = 356 difference: 8796 2 pieces conclusion: TP Chimeric read: a0c7035c-ee8a-42d6-a65c-587ea96031bd_Basecall_1D_template 4351214 4351239 ref distance: 25 read distance: 1633-1632 = 1 difference: 24 2 pieces conclusion: TP Chimeric read: fabd41fd-e499-441e-84f8-a0924740cfe7_Basecall_1D_template 3598627 3414887 ref distance: 183740 read distance: 1354-1353 = 1 difference: 183739 2 pieces conclusion: TP Chimeric read: 0afb4f6f-d60d-4099-80b8-668056d4f9d2_Basecall_1D_template 756616 3114568 ref distance: 2357970 read distance: 176-171 = 5 difference: 2357965 2 pieces conclusion: TP  Results from metagenome run (ActinoMock X0163): /global/projectb/scratch/ashleigh/chimeric/X0163/X0163_small.fastq 500,000 reads 10,337 are chimeric

Chimeric read: b5039ed2-a9b3-4d9c-a435-c58a95055ea2_Basecall_1D_template Position 1: 165022 Position 2: 3025296 Reference distance: 2860274 Read distance: 3539-3510 = 29 Difference: 2860245 Number of pieces: 2 Conclusion: TP Chimeric read: bbcc16f5-5257-4dd3-9632-f6a7ed614886_Basecall_1D_template Position 1: 2877556 Position 2: 255 Reference distance: 2877301 Read distance: 1282-1251 = 31 Difference: 2877270 Number of pieces: 2 Conclusion: TP Chimeric read: 093571b1-d3a2-45d6-8ed2-db69ab472ced_Basecall_1D_template Position 1: 2643725 Position 2: 171207 Reference distance: 2472518 Read distance: 885-824 = 61 Difference: 2472457 Number of pieces: 2 Conclusion: TP Chimeric read: d8b2fb8e-0476-4f64-af71-a726a9ac9011_Basecall_1D_template Position 1: 1044000 Position 2: 255 Reference distance: 1043745 Read distance: 1337-1311 = 26 Difference: 1043719 Number of pieces: 2 Conclusion: TP Chimeric read: 61437a9a-e2b9-417e-99e0-02c809711586_Basecall_1D_template Position 1: 625910 Position 2: 1149771 Reference distance: 523861 Read distance: 1705-1655 = 50 Difference: 523811 Number of pieces: 2 Conclusion: TP Chimeric read: 11886edb-a844-47df-8f5e-8519d8d32ffb_Basecall_1D_template Position 0: 1844594 (AM 8) Position 1: 3386543 (AM 3) Position 2: 1327974 (AM3) Reference distance: Read distance:1502-1499 = 3 Difference: Number of pieces: 3 Conclusion: TP, definitely chimeric Chimeric read: 7e7a9ba6-09f8-49c8-aa61-92410595f534_Basecall_1D_template Position 1: 3990998 Position 2: 1816449 Reference distance: 2174549 Read distance:3532-3469=63 Difference: 2174486 Number of pieces:2 Conclusion: TP Chimeric read: 1c423dd6-5db1-4ff5-879b-f37a7c9e0a37_Basecall_1D_template Position 0: 537113 AM1a Position 1: 254262 AM1a Position 2: 229153 AM2 Reference distance: Read distance: Difference: Number of pieces: 3 Conclusion: TP Chimeric read: 92468353-8707-4c6d-babd-2e659da427ca_Basecall_1D_template Position 1: 2655468 Position 2: 3456517 Reference distance: 801049 Read distance: 3289-3287 = 2 Difference: 801047 Number of pieces: 2 Conclusion: TP Chimeric read: c537fffe-cd6d-4558-8421-2e2247364ccc_Basecall_1D_template Position 1: 336712 Position 2: 332822 Reference distance: 3890 Read distance: 2674-2646 = 28 Difference: 3862 Number of pieces: 2 Conclusion: TP Chimeric read: e014d7b6-8178-4d36-9267-39b6edf68e40_Basecall_1D_template Position 1: 1067315 Position 2: 5289438 Reference distance: 4222123 Read distance: 799-744 = 55 Difference: 4222068 Number of pieces: 2 Conclusion: TP Chimeric read: 069bae3d-0ec3-40f8-83aa-06d02639db7d_Basecall_1D_template Position 1: 879318 Position 2: 881974 Reference distance: 2656 Read distance:4362-2513 = 1849 Difference: 807 Number of pieces: 2 Conclusion: TP Chimeric read: 9643e482-426d-4c38-a3da-f77a8dd64a9f_Basecall_1D_template Position 1: 2391165 Position 2: 1890673 Reference distance: 500492 Read distance: 3015-2990 = 25 Difference: 500467 Number of pieces: 2 Conclusion: TP Chimeric read: a04f87cc-732d-4d70-9214-4db7c35aafda_Basecall_1D_template Position 1: 1392146 Position 2: 1392139 Position 3: 3649554 Reference distance: 2257415 ? (3-1) Read distance: 5023-1918 = 3105 Difference: 2254310 Number of pieces: 2 with small gap Conclusion: TP, probably just a gap Chimeric read: 50ef1361-a8be-40bd-8fc6-d05fc7f88ada_Basecall_1D_template Position 1: 2744519 Position 2: 1020564 Reference distance: 1723955 Read distance:12712-12592 = 120 Difference: 1723835 Number of pieces: 2 Conclusion: TP Chimeric read: 3406624d-aef2-49ca-8f50-2c23bbed7032_Basecall_1D_template Position 1: 707114 Position 2: 2069936 Reference distance: 1362822 Read distance:3469-3415 = 54 Difference: 1362768 Number of pieces: 2 Conclusion: TP Chimeric read: b7e8c4b5-1fbc-491a-b491-de1419f65b58_Basecall_1D_template Position 1: 2516837 Position 2: 1127938 Reference distance: 1388899 Read distance:894-835 = 59 Difference: 1388840 Number of pieces:2 Conclusion: TP Chimeric read: 04cef3b5-581b-41b1-b744-bd363f426747_Basecall_1D_template Position 1: 770492 Position 2: 2031873 Reference distance: 1261381 Read distance:1517-1461=56 Difference: 1261325 Number of pieces:2 Conclusion: TP Chimeric read: 97aaf4df-23de-4f4f-bd6a-91451b24491a_Basecall_1D_template Position 1: 348181 Position 2: 1177910 Reference distance: 829729 Read distance:2125-2121=4 Difference: 829725 Number of pieces:2 Conclusion: TP Chimeric read: 1225c4bf-7c20-49e8-9695-d0d75a3ceec4_Basecall_1D_template Position 1: 2227483 Position 2: 1552265 Reference distance: 675218 Read distance:1785-1708=77 Difference: 675141 Number of pieces:2 Conclusion: TP Analysis of Not_covered in X0163 (metagenome) c5f22007-690c-4ecc-bf7e-e03acba85c59_Basecall_1D_template Reference mapping: 0 a392a9d7-d46a-465f-895a-e072d6b7222d_Basecall_1D_template Reference mapping: 0 2743687f-6256-4e93-a81a-fc2896df76f8_Basecall_1D_template: Reference mapping: 0 4ef27459-592c-4a78-a5db-1c58205eb841_Basecall_1D_template Reference mapping: 0 1c82aedb-9913-493b-a4b2-11a9ff19e158_Basecall_1D_template Reference mapping: 0 d89a2cc2-9009-4de6-85a4-5a10d5502c88_Basecall_1D_template Reference mapping: 0 b3e41bf7-3dce-4be1-a122-5002e4bf4a3b_Basecall_1D_template Reference mapping: 0 89809a7a-95d5-4434-b6ab-9f50361138e2_Basecall_1D_template Reference mapping: 0 4b5ba5ed-68c8-44e4-963d-8ec4e2c85928_Basecall_1D_template Reference mapping: 0 8c587974-2d8e-4ad8-82a4-08c326833ded_Basecall_1D_template Reference mapping: 0 2005fc05-4e15-4b40-9d39-3847b012e012_Basecall_1D_template Reference mapping: 0 a7eb81ba-2f89-44cd-afd3-afa1a03dd716_Basecall_1D_template Reference mapping: 0 b29f43f6-7c96-45d2-81ea-26e0b18d69e5_Basecall_1D_template Reference mapping: 4392406 88b6e8dd-5845-4702-9bff-1428784f0131_Basecall_1D_template Reference mapping: 0 90ef8a26-419c-4cf0-9fb5-169c8a0c54c2_Basecall_1D_template Reference mapping: 0 e59ac1c6-e0f9-485b-b8b0-7fd85ba30b87_Basecall_1D_template Reference mapping: 0 47103d13-46a9-4f27-afe5-d88c521c1414_Basecall_1D_template Reference mapping: 0 f6dacf34-00c3-433c-bbc0-d031a18858a1_Basecall_1D_template Reference mapping: 0 9ad90ff1-dd36-4e51-b7e9-57490f8694b0_Basecall_1D_template Reference mapping: 0